Treatment of amyotrophic lateral sclerosis (als)

ABSTRACT

The present disclosure relates to AAVs encoding a SOD1 targeting polynucleotide which may be used to treat amyotrophic lateral sclerosis (ALS) and delivery methods for the treatment of spinal cord related disorders including ALS.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Provisional Patent ApplicationNo. 62/572,702, filed Oct. 16, 2017, Provisional Patent Application No.62/578,735, filed Oct. 30, 2017, and Provisional Patent Application No.62/666,934, filed May 4, 2018, all entitled Treatment of AmyotrophicLateral Sclerosis (ALS); the contents of each of which are hereinincorporated by reference in their entirety.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format as an ASCII text file. The Sequence Listing isprovided as an ASCII text file entitled 20571055PCT_SEQLST.txt, createdon Oct. 16, 2018, which is 7,474 bytes in size. The Sequence Listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to compositions, methods and processes forthe design, preparation, manufacture and/or formulation ofpolynucleotides, including AAV vectors, small interfering RNA (siRNA)duplexes, shRNA, microRNA or precursors thereof which target or encodemolecules which target the superoxide dismutase I (SOD1) gene tointerfere with SOD1 gene expression and/or SOD1 enzyme production. Insome embodiments, polynucleotides are inserted into recombinantadeno-associated virus (AAV) vectors.

Methods for inhibiting SOD1 or altering the expression of any geneassociated with a spinal cord related disease or disorder in a subjectwith a disease and/or other disorder associated with the spinal cord arealso disclosed. The method includes the administration of the at leastone polynucleotide into the subject with a disorder associated with thespinal cord (e.g., neurodegenerative disease) via at least the rought ofintraparenchymal delivery to the spinal cord. In these embodiments thedisease is a motor neuron disease, and more specifically, the disease isamyotrophic lateral sclerosis (ALS).

BACKGROUND OF THE INVENTION

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease,is a fatal progressive neurodegenerative disease, characterized by thepredominant loss of motor neurons (MNs) in primary motor cortex, thebrainstem, and the spinal cord. The loss of motor neurons devastatesbasic fundamental movements, such as breathing, and typically causesdeath to patients within 2-5 years after diagnosis. Progressivedeterioration of motor function in patients severely disrupts theirbreathing ability, requiring some fbrm of breathing aid for survival ofthe patients. Other symptoms also include muscle weakness in hands,arms, legs or the muscles of swallowing. Some patients (e.g., FTD-ALS)may also develop frontotemporal dementia.

According to the ALS Association, approximately 5,600 people in theUnited States of America are diagnosed with ALS each year. The incidenceof ALS is two per 100,000 people, and it is estimated that as many as30,000 Americans may have the disease at any given time.

Two forms of ALS have been described: one is sporadic ALS (sALS), whichis the most common form of ALS in the United. States of America andaccounts for 90 to 95% of all cases diagnosed; the other is familial ALS(fALS), which occurs in a family lineage mainly with a dominantinheritance and only accounts for about 5 to 10% of all cases in theUnited States of America. sALS and fALS are clinicallyindistinguishable.

Pathological studies have linked numerous cellular processes with thedisease pathogenesis such as increased ER stress, generation of freeradicals (i.e., reactive oxygen species (ROS)), mitochondria'dysfunction, protein aggregation, apoptosis, inflammation and glutamateexcitotoxicity, specifically in the motor neurons (MNs).

The causes of ALS are complicated and heterogeneous. In general, ALS isconsidered to be a complex genetic disorder in which multiple genes incombination with environmental exposures combine to render a personsusceptible. More than a dozen genes associated with ALS have beendiscovered, including, SOD1 (Cu²⁺/Zn²⁺ superoxide dismutase), TDP-43(TARDBP, TAR DNA binding protein-43), FUS (Fused in SarcomaiTranslocatedin Sarcoma), ANG (Angiogenin), ATXN2 (Ataxin-2), valosin containingprotein (VCP), OPTN (Optineurin) and an expansion of the noncodingGGGGCC hexanucleotide repeat in the chromosome 9, open reading frame 72(C9ORF72), However, the exact mechanisms of motor neuron degenerationare still elusive.

Currently, there is no curative treatment for ALS. Until recently, theonly FDA approved drug was Riluzole, which antagonizes the glutamateresponse to reduce the pathological development of ALS. However, onlyabout a three-month life span expansion for ALS patients in the earlystages has been reported, and no therapeutic benefit for this patientpopulation (Bensimon G et al. J Neural. 2002, :249, 609-615). In 2017,the FDA approved Ra.dicava. (edaravone) for the treatment of ALS, thefirst of such approval in 22 years. Radicava is administeredintravenously and serves as a free-radical scavenger, reducing oxidativestress in patients suffering from ALS and thereby slowing diseaseprogression. In a clinical Phase 3 trial (NCT01492686) of 137 patients,Radicava slowed the decline in physical ⁻function as compared to thosepatients taking placebo and as determined by score on the ALS FunctionalRating Scale-Revised (ALSFRS-R) (Writing group; Edaravone (MCI-186) ALS19 Study Group Lancet Neurol, 2017 July; 16(7):505-512). The approval ofRadicava is considered an advance in terms of treatment of ALS, howeverit is still not a cure. New treatment strategies that can effectivelyprevent and/or significantly hinder the disease progression are still indemand.

Mutations in the gene of Cu²⁺/Zn²⁺ superoxide dismutase type I (SOD1)are the most common cause of fALS, accounting for about 20 to 30% of allfALS cases. Recent reports indicate that SOD1 mutations may also belinked to about 4% of all sALS cases (Robberecht and Philip, Nat. Rev.Neurosci., 2013, 14, 248-264), SOD1-linked fALS is most likely notcaused by loss of the normal SOD1 activity, but rather by a gain of atoxic function. One of the hypotheses fbr mutant SOD1-linked fALStoxicity proposes that an aberrant SOD1 enzyme causes small moleculessuch as peroxynitrite or hydrogen peroxide to produce damaging freeradicals. Other hypotheses for mutant SOD1 neurotoxicity includeinhibition of the proteasome activity, mitochondrial damage, disruptionof RNA processing and formation of intracellular aggregates. Abnormalaccumulation of mutant SOD1 variants and/or wild-type SOD1 in ALS formsinsoluble fibrillar aggregates which are identified as pathologicalinclusions. Aggregated SOD1 protein can induce mitochondria stress(Vehvilainen P et al., Front Cell Neurosci., 2014, 8, 126) and othertoxicity to cells, particularly to motor neurons.

These findings indicate that SOD1 can be a potential therapeutic targetfor both familial and sporadic ALS. A therapy that can reduce the SOD1protein, whether wildtype or mutant, produced in the central nervoussystem of ALS patients may ameliorate the symptoms of ALS in patientssuch as motor neuron degeneration and muscle weakness and atrophy.Agents and methods that aim to prevent the formation of wild type and/ormutant SOD1 protein aggregation may prevent disease progression andallow for amelioration of ALS symptoms. RNA interfering (RNAi) mediatedgene silencing has drawn researchers' interest in recent years, Smalldouble stranded RNA (small interfering RNA) molecules that target SOD1gene have been taught in the art for their potential in treating ALS(See, e.g., U.S. Pat. No. 7,632,938 and U.S. Patent Publication No.20060229268).

The present disclosure develops an RNA interference or knock-down based.approach to inhibit or prevent the expression of SOD1 gene in ALSpatients for treatment of disease.

The present disclosure provides novel polynucleotides, including doublestranded RNA (d.sRNA) constructs and/or siRNA constructs, shRNAconstructs and/or micmRNA constructs and methods of their design. Inaddition, these siRNA constructs may be synthetic molecules encoded inan expression vector (one or both strands) for delivery into cells. Suchvectors include, but are not limited to adeno-associated viral vectorssuch as vector genomes of any of the AAV serotypes or other viraldelivery vehicles such as lentivirus, etc.

The present disclosure also provides novel methods for the deliveryand/or transmission of the AAV vectors and viral genomes of thedisclosure, which may be applied to other disorders associated with thespinal cord, such as, but not limited to, the larger family of motorneuron disorders, neuropathies, diseases of myelination, andproprioceptive, somatosensory and/or sensory disorders.

SUMMARY OF THE INVENTION

The present disclosure provides AAV vectors encoding a SOD1 targetingpolynucleotide to interfere with SOD1 gene expression and/or SOD1protein production and methods of use thereof. Methods for treatingdiseases associated with motor neuron degeneration such as amyotrophiclateral sclerosis are also included in the present disclosure.

In some embodiments, the present disclosure provides a method forinhibiting the expression of the SOD1 gene in a subject which mayinvolve administering to the subject a composition comprising an AAVvector at one or more sites by intraparenchymal delivery. The AAV vectorcomprises a vector genome and a capsid, and the vector genome includes amodulatory polynucleotide sequence positioned between two invertedterminal repeats (ITRs). In some embodiments, administration may occurat one or more sites within the spinal cord of the subject. In someembodiments, administration may occur at two sites within the spinalcord of the subject.

Also provided herein is a method for treating and/or amelioratingamyotrophic lateral sclerosis (ALS) in a subject which may involveadministering to the subject in need of treatment a therapeuticallyeffective amount of a composition comprising an AAV vector at one ormore sites by intraparenchymal delivery. The AAV vector comprises avector genome and a capsid, and the vector genome includes a modulatorypolynucleotide sequence positioned between two inverted terminal repeats(JIRO. in some embodiments, the expression of SOD/ is inhibited orsuppressed. In some embodiments, administration may occur at one or moresites within the spinal cord of the subject. In some embodiments,administration may occur at two sites within the spinal cord of thesubject.

The SOD1 gene may be wild type SODI, mutated SOD1 with at least onemutation or both wild type SOD1 and mutated SOD/ with at least onemutation. In some embodiments, the expression of SOD1 is inhibited orsuppressed by about 20% to about 100%.

In some embodiments, the method may involve administering thecomposition by intraparenchymal delivery at two sites at the cervicalspinal cord region. In one embodiment, the method may involveadministering intraparenchymally to the levels C3 and C5.

In some embodiments, the volume of administration is 5 μL to 240 μL atlevel C3 of the spinal cord and 5 μL to 240 μL at level C5 of the spinalcord. In some embodiments, the volume of administration is 5 μL to 60 μLat level C3 of the spinal cord and 5 μL to 60 μL at level C5 of thespinal cord. In some embodiments, the volume of administration is 25 to40 μL at level C3 of the spinal cord and 25 to 40 μL at level C5 of thespinal cord. In some embodiments, the dose is 1×10¹⁰ vg to 1×10¹² vg atlevel C3 of the spinal cord and 1×10¹⁰ vg to 1×10¹⁷ vg at level C5 ofthe spinal cord. In some embodiments, the dose is 5×10¹¹vg to 8×10¹¹ vgat level C3 of the spinal cord and 5×10¹¹ vg to 8×10¹¹ vg at level C5 ofthe spinal cord. In some embodiments, the composition is administered ata rate of 5 μL/min.

In other embodiments, the method may involve administering thecomposition intraparenchymally to any two sites selected from C1, C2,C3, C4, C5, C6, and C7.

In some embodiments, the method may involve administering thecomposition by intraparenchymal delivery at two sites at the thoracicspinal cord region. In some embodiments, the two sites at the thoracicspinal cord region may be at any two levels selected from T1, T2, T3,T4, T5, T6, T7, T8, T9, T10, T11, and T12.

In some embodiments, the method may involve administering thecomposition by intraparenchymal delivery at two sites at the thoracicspinal cord region. In some embodiments, the two sites at the lumbarspinal cord region may be at any two levels selected from L1, L2, L3,L4, and L5.

In other embodiments, the method may involve administering thecomposition by intraparenchymal delivery to one or more regions selectedfrom the cervical spinal cord, thoracic spinal cord, lumbar spinal cord,and sacral spinal cord. In some embodiments, such method may involveadministering intraparenchymally to one or more sites independentlyselected from C1, C2, C3, C4, C5, C6, C7, T1, T2, T3, T4, T5, T6, T7,T8, T9, T10, T11, T12, L1, L2, L3, L4, and L1. In some embodiments, suchmethod may involve administering intraparenchymally to one or more sitesindependently selected from C1, C2, C3, C4, C5, C6, C7, and L1,

In one embodiment, SOD1 is suppressed 30% in a subject treated with anAAV encoding a SOD1 targeting polynucleotide as compared to an untreatedsubject. The subject may be administered the AAV in an infusion or as abolus at a pre-determined dose level. As a non-limiting example, thesuppression is seen in the C 1 to L7 ventral horn region.

The present disclosure relates to RNA molecule mediated gene specificinterference with gene expression and protein production. Methods fortreating diseases associated with motor neuron degeneration such asamyotrophic lateral sclerosis are also included in the presentdisclosure. The siRNA included in the compositions featured hereinencompass a dsRNA having an antisense strand (the antisense or guidestrand) having a region that is 30 nucleotides or less, generally 19-24nucleotides in length, that is substantially complementary to at leastpart of an mRNA transcript of the SOD1 gene.

The present invention provides short double stranded RNA molecules suchas small interfering RNA (siRNA) duplexes that target SOD1 mRNA tointerfere with SOD1 gene expression and/or SOD1 protein production. ThesiRNA duplexes of the present invention may interfere with both allelesof the SOD1 gene irrespective of any particular mutation in the SOD1gene, and may particularly interact with those found in ALS disease.

In some embodiments, such siRNA molecules, or a single strand of thesiRNA molecules, are inserted into adeno-associated viral vectors to beintroduced into cells, specifically motor neurons and/or othersurrounding cells in the central nervous system.

The siRNA. duplex of the present invention comprises an antisense strandand a sense strand hybridized together forming a duplex structure,wherein the antisense strand is complementary to the nucleic acidsequence of the targeted SOD1 gene, and wherein the sense strand ishomologous to the nucleic acid sequence of the targeted SOD1 gene. Insome aspects, the 5′end of the antisense strand has a 5′ phosphate groupand the 3′end of the sense strand contains a 3′hydroxyl group, In otheraspects, there are none, one or 2 nucleotides overhangs at the 3′end ofeach strand.

According to the present disclosure, each strand of the siRNA duplextargeting the SOD1 gene is about 19-25 nucleotides in length, preferablyabout 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23nucleotides, 24 nucleotides, or 25 nucleotides in length. In someaspects, the siRNAs may be unmodified RNA molecules.

In one embodiment, an siRNA or dsRNA includes at least two sequencesthat are complementary to each other. The dsRNA includes a sense strandhaving a first sequence and an antisense strand having a secondsequence. The antisense strand includes a nucleotide sequence that issubstantially complementary to at least part of an mRNA encoding SOD1,and the region of complementarity is 30 nucleotides or less, and atleast 15 nucleotides in length. Generally, the dsRNA is 19 to 24, e.g.,19 to 21 nucleotides in length. n some embodiments the dsRNA is fromabout 15 to about 25 nucleotides in length, and in other embodiments thedsRNA is from about 25 to about 30 nucleotides in length.

In one embodiment, the sense strand sequence comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from thenucleotide sequence of SEQ ID NO: 7 and the antisense strand sequencecomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO: 8.

The dsRNA, either upon contacting with a cell expressing SOD1 or upontranscription within a cell expressing SOD1, inhibits or suppresses theexpression of a SOD1 gene by at least 10%, at least 20%, at least 25%,at least 30%, at least 35% or at least 40% or more, such as when assayedby a method as described herein.

According to the present disclosure, AAV vectors comprising the nucleicacids encoding the siRNA duplexes, one strand of the siRNA duplex or thedsRNA targeting SOD1 gene or other neurodegenerative associated gene orspinal cord disease associated gene are produced, the AAV vectorserotype may be AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4,AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1 .2, AAV7, AAV7.2, AAV8, AAV9,AAV9.11AAV9,13, AAV9.16, AAV9.24, AAV9.45A,AV9,47, AAV9.61, AAV9.68,AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3,AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a,AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13,AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23,AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223,2,AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7/rh.48, AAV 1-8/rh.49,AAV2-15/rh.62, AAV2-3/rh.61, AAV2-4/rh.50, AAV2-5/rh, 51, AAV3.1/hu.6,AAV3.1/hu.9, AAV3-9/rh.52, AAV3-11/rh.53, AAV4-8/r11.64, AAV4-9/rh.54,AAV4-19/rh.55, AAV5-3/rh.57, AAV5-22/rh,58, AAV7.3/hu.7, AAV16.8/hu.10,AAV16.12/hu.11, AAV29.3/bb.1, AAV29.5/bb.2, AAV106.1/hu.37,AAV114.3/hu.40, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.3/hu.44,AAV130.4/hu.48, AAV1.45.1./hu.53, AAV 145.5/hu.54, AAV145.6/hu.55,AAV161.10/hu.60, AAV161.6/hu.61, AAV33.12/hu.17, AAV33.4/hu.15,AAV33.8/hu.16, AAV52/hu.19, AAV52.1/hu.20, AAV58.2/hu.25, AAVA3.3,AAVA3.4, AAVA3.5, AAVA3.7, AAVC1, AAVC2, AAVC5, AAV-DJ, AAV-DJ8, AAVF3,AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi.1, AAVpi.3,AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69,AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVLK03, AAVH-1/hu.1, AAVH-5//hu.3,AAVLG-10/rh.40, AAVLG-4/rh.38, AAVLG-9/hu.39, AAVN721-8/rh,43, AANCh.5,AAVCh.5R1, AAVcy.2, AAVcy.3, AANcy.4, AAVcy.5, AAVCy.5R.1, AAVCy.5R2,AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4,AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.11, AAVhu.13,AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.20, AAVhu.21, AAVhu.22,AAVhu.23.2, AA.Vhu,24, AAVhu.25, AAVItu.27, AAVItu.28, AAVhu.29,AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39,AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44R1,AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AA.Vhu,47, AA.Vhu,48,AA.Vhu.48R1, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52,AAVhu,54, AAVhu,55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61,AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14/9, AAVhu.t 19, AAVrh.2,AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R,AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22,AAVrh.23, AAVrh.24, AAVrh.2.5AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34,AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40,AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49,AAVrh.51, AAVrh,52, AAVrh.53, AAVrh.54, AAVrh.56AAVrh.57, AAVrh.58,AAVrh.61, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73,AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, AAAV,BAAV, caprine AAV, bovine AAV AAVhE1.1, AAVhEr1.5, AAVhER1.14,AAVhEr1.8, AAVhEr1.16, AAVhEr1.18, AAVhEr1.35AAVhEr1.7, AAVhEr1.36,AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36,AAVhER.1.23, AAVhEr3.1, AAV2.5T , AAV-PAEC, AAV-LK01, AAV-LK02,AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09,AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16,AAV-LK17AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7,AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b,AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAVShuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAVShuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10,BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48,AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8/rh.64, AAVLG-9/hu.39,AAV54.5/hu.23, AAV54.2/hu.22, AAV54.7/hu.24, AAV54.1/hu.21,AAV54.412/hu.27, AAV46.2/hu.28, AAV46.6/hu.29, AAV1.28.1/hu.43, truetype AAV (ttAAV), UPENN AAV 10, Japanese AAV 10 serotypes, AAV CBr-7.1,AAV CBr-7.10, AAV CBr-7.2, AAV CBr-73, AAV CBr-7.4, AAV CBr-7.5, AAVCBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-E1AAV CBr-E2,AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7,AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10,AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-P1, AAVCHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAVCKd-10, AAV CKd-7 AAV CKd-3, AAV CKd-44-AV CKd-6, AAV CKd-7, AAV CKd-8,AAV CKd-B1, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6,AAV CKd-B7, AAV CKd-B8, AAV CKd-H1, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4,AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-F1,AAV CLg-F2, A-AV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7,AAV CLg-F8, AAV CI-v-1, AAV CLv1-1, AAV Clv1-10, AAV CLv1-2, AAV CLv-12,AAV CLv1-3, AAV CLv-13, AAV CLv1-4, AAV Clv1-7, AAV Clv1-8, AAV Clv1-9,AAV CLv-2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-D1, AAVCLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAVCLv-D8, AAV CLv-E1, AAV CLv-K1, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAVCLv-L5, AAV CLv-L6, AAV CLv-M1, AAV CLv-M11, AAV CLv-M2, AAV CLv-M5, AAVCLv-M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV Chv-R1, AAV CLv-R2, AAVCLv-R3, AAV CLv-R4, AAV CLv-R5,AAV CLV-R6, AAV CLv-R7, AAV CLv-R8, AAVCLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAVCSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.10, AAV CSp-8.2, AAVCSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAVCSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5,AAVF1/HSC 1, AAVF11/HSC11, AAVF12/HSC12, AAVF13/HSC 13, AAVF14/HSC14,AAVF15/HSC15, AAVF16/HSC16, AAVF17/HSC17, AAVF2IHSC2, AAVF3/HSC3AAVF4/HSC4, AAVF5/HSC5, AAVF6/HSC6, AAVF7/HSC7, AAVF8/HSC8, AAVF9/HISC9,AAV-PHP.B, AAV-PHP.A, G2B-26, G2B-13, TH1.1-32, TH1.1-35, AAVPHP.B2,AAVPHP.B3, AAVPHP.N/PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT,AA.VPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T AAVPHP.B-GGT-T,AAVPHIP.B-SGS, AAVPH.P.B-AQP, AAVPHP.B-QQP, AAVPHIP.B-SNP(3),AAVPHIP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN,AAVPHP.B-EGTNAVPHRB-DST, AAVPHP.B-DST. AAVPHP.B-STP, AA.VPHP.B-PQP,AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, A,AVPHP.B-TTP, AAVPHP.S/G2A12,AAVG2A15/G2A3, AAVG2B4, AAVG2B5 and variants thereof.

In one embodiment, the AAV vector serotype is AAVrh.10 or a variantthereof.

According to the present invention, siRNA duplexes or dsRNA targetingthe SOD1 gene in ALS are selected from the siRNA duplexes listed inTable 4. In one aspect, the siRNA duplexes or dsRNA targeting SOD1 genein ALS is D-4012.

The present disclosure also provides pharmaceutical compositionscomprising at least one siRNA duplex targeting the SOD1 gene and apharmaceutically acceptable carrier. In some aspects, a nucleic acidsequence encoding the siRNA duplex is inserted into an AAV vector.

In some embodiments, the present disclosure provides methods forinhibiting/silencing of SOD1 gene expression in a cell. Accordingly, thesiRNA duplexes or dsRNA can be used to substantially inhibit SOD1 geneexpression in a cell, in particular in a motor neuron. In some aspects,the inhibition of SOD1 gene expression refers to an inhibition by atleast about 20%, preferably by at least about 30%, 40%, 50%, 60%, 70%,80%, 85%, 90%, 95% and 100%. Accordingly, the protein product of thetargeted gene may be inhibited by at least about 20%, preferably by atleast about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. TheSOD1 gene can be either a wild type gene or a mutated SOD1 gene with atleast one mutation. Accordingly, the SOD1 protein is either wild typeprotein or a mutated polypeptide with at least one mutation.

In some embodiments, the present disclosure provides methods fortreating, or ameliorating amyotrophic lateral sclerosis associated withabnormal SOD1 gene and/or SOD1 protein in a subject in need oftreatment, the method comprising administering to the subject apharmaceutically effective amount of at least one siRNA duplex targetingthe SOD1 gene, delivering said siRNA duplex into targeted cells,inhibiting SOD1 gene expression and protein production, and amelioratingsymptoms of ALS in the subject.

In some embodiments, the AAV vector genome may include a promoter. Inone aspect, the promoter may be H1.

In some embodiments, an AAV vector comprising the nucleic acid sequenceencoding at least one siRNA duplex targeting the SOD1 gene isadministered to the subject in need for treating and/or amelioratingALS. The AAV vector serotype may be selected from the group consistingof AAV1, AAA 2, AAV2G9, AAV3, AAV3a, AAV3b.NAV3-3NAV4, AAV4-4, AAV5,AAV6, AAV6.1, AAV6.2, A.AV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11,AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84,AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12,AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b,AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15,AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25,AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1., AAV223.2, AAV223.4,AAV223.5, AAV223.6, AAV223.7, AAV1-7/rh.48, AAV1-8/rh.49, AAV2-15/rh,62,AAV2-3/rh.61, AAV2-4/rh.50, AAV2-5/rh.51, AAV3.1/hu.6, AAV3.1/hu.9,AAV3-9/rh.52, AAV3-11/rh.53, AAV4-8/r11.64, AAV4-9/rh.54, AAV4-19/rh.55,AAV5-3/rh.57, AAV5-22/rh.58, AAV7.3/hu.7, AAV1.6.81hu.10,AAV16.12./hu.11, AAV29.3/bb.1, AAV29.5/bb.2, AAV106.1/hu.37,AAV114.3/hu,40, AAV127,2/1m.41, AAV127.5/hu,42, AAV128,3/hu.44,AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54, AAV145.6/hu.55,AAV161.10/hu.60, AAV161.6/hu.61, AAV33.12/hu.17, AAV33.4/hu.15,AAV33.81hu.16, AAV52/hu.19, AAV52.1/hu.20, AAV58.2/hu.25, AAVA3.3,AAVA3.4, AAVA3.5, AAVA3.7, AAVC1, AAVC2, AAVC5, AAV-DJ, AAV-DJ8, AAVF3,AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVr11.70, AA,Vpi.1, AAVpi.3,AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVeh.69,AAVrh.45, AAVrh,59, AAVhu.12, AAVH6, AAVLK03, AAVH-1./hu.1, AAVH-5/hu.3,AAVLG-10/rh.40, AAVLG-4/rh.38AAVLG-9/hu.39, AAVN721-8/rh.43, AAVCh.5,AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1, AAVCy.5R2,AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4,AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.11, AAVhu.13,AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.20, AAVhu.21, AAVhu.22,AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R,AAVhu.31, AAVhu.32, AAVhu.34. AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40,AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44R1, AAVhu.44R2,AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48R1,AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54,AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63,AAVhu.64, AAVhu.66, AAVhu.67, AAhu.14/9, AAVhu.t19. AAVrh.2, AAVrh.2R,AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14,AAVrh.17, AAAVrh.18, AVrh.19, AAVrh.20, AAVrh.21., AAVrh.22, AAVrh.23,AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35,AAVrh,36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46,AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51,AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61,AAVrh.64, AAVrh.64R1. AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R,AAVrh8R A586R mutant. AAVrh8R R533A mutant, AAAV, BAAV, caprine AAV,bovine AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr.1.8, AAVhEr1.16,AAVhEr1.18, AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4,AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1,AAV2.5T , AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05,AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12,AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19,AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11,AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h NAV-8b, AAV-h, AAV-h, AAV-b, AAVSM 10-2, Shuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAVShuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAVSM 104, AAV SM 10-8 , AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV,BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11,AAVhu.53, AAV4-8/rh.64, AAVLG-9/hu.39, AAV54.5/hu.23, AAV54.2/hu.22,AAV54.7/hu.24, AAV54.1/hu.21, AAV54.4R/hu.27, AAV46.2/hu.28,AAV46.6/hu.29, AAV128.1/hu.43, true type AAV (ttAAV), UPENN AAV 10,Japanese AAV 10 serotypes, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAVCBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7,7, AAV CBr-7.8, AAVCBr-B7.3, AAV CBr-B7.4, AAV CBr-E1, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4,AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1,AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAVCHt-6.6, AAA CHt-6.7, AAV CHt-6.8, AAV CHt-P1, AAV CHt-P2, AAV CHt-P5,AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2,AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-B1, AAVCKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, , AAV CKd-B7,AAA CKd-B8, AAA CKd-H1, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5,AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-F1, AAV CLg-F2,AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8,AAV CLv-1, AAV CLv1-1, AAV Clv1-10, AAV CLv1-2, AAV CLv-12, AAV CLv1-3,AAV CLv-13, AAV CLv1-4, AAV Clv1-7, AAV Clv1-8, AAV C1v1-9, AAV CLv-2,AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV Clv-D1, AAV CLv-D2, AAVCLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, ,AAV CLv-E1, AAV CLv-K1, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5,AAV CLv-L6, AAV CLv-M1, AAV CLv-M11, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6,AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-R1, AAV CLv-R2, AAV CLv-R3,AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9,AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAVCSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.10, AAV CSp-8.2, AAV CSp-8.4, AAVCSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9,AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1/HSC1, AAVF11/HSC11,AAVF12/HSC12, AAVF1.3/HSC13, AAVF14/HSC14, AAVF15/HSC15, AAVF16/HSC16,AAVF17/HSC17, AAVF2/HSC2, AAVF3/HSC3, AAVF4/HSC4, AAVF5/HSC5,AAVF6/HSC6, AAVF7/HSC7, AAVF8/HSC8, AAVF9/HSC9, AAV-PHP.B, AAV-PHP.A,G2B-26, G2B-13, TH1.1-32, TH1.1-35, AAVPHP.B2, AAVPHP.B3,AAVPH.P.N/PHIP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP,AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS,AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT,AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST,AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHR.B-SQP, AAVPHP.B-QLP,AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S/G2A12, AAVG2A15/G2A3, AAVG2B4,AAVG2B5 and variants thereof.

In some aspects, ALS is familial ALS linked to SOD1 mutations. In otheraspects, ALS is sporadic ALS which is characterized by abnormalaggregation of SOD1 protein or disruption of SOD1 protein function orlocalization, though not necessarily as a result of genetic mutation.The symptoms of ALS ameliorated by the present method may include motorneuron degeneration, muscle weakness, stiffness of muscles, slurredspeech and/or difficulty in breathing.

In some embodiments, the siRNA duplexes or dsRNA targeting SOD1 gene orthe AAV vectors comprising such siRNA-encoding molecules may beintroduced directly into the central nervous system of the subject, forexample, by intracranial injection.

In some embodiments, the pharmaceutical composition of the presentdisclosure is used as a solo therapy. In other embodiments, thepharmaceutical composition of the present disclosure is used incombination therapy. The combination therapy may be in combination withone or more neuroprotective agents such as small molecule compounds,growth factors and hormones which have been tested for theirneuroprotective effect on motor neuron degeneration.

In some embodiments, the present disclosure provides methods fortreating, or ameliorating amyotrophic lateral sclerosis by administeringto a subject in need thereof a therapeutically effective amount of aplasmid or AAV vector described herein. The ALS may be familial ALS orsporadic ALS.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to SOD1 targeting polynucleotides astherapeutic agents. RNA interfering mediated gene silencing canspecifically inhibit gene expression. The present disclosure thereforeprovides polynucleotides such as small double stranded RNA (dsRNA)molecules (small interfering RNA, siRNA), shRNA, microRNA and precursorsthereof targeting SOD1 gene, pharmaceutical compositions encompassingsuch polynucleotides, as well as processes of their design. The presentdisclosure also provides methods of their use for inhibiting SOD1 geneexpression and protein production, for treating disorders associatedwith the spinal cord and/or neurodegenerative disease, in particular,amyotrophic lateral sclerosis (ALS).

The details of one or more embodiments of the disclosure are set forthin the accompanying description below. Although any materials andmethods similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, the preferredmaterials and methods are now described. Other features, objects andadvantages of the disclosure will be apparent from the description. inthe description, the singular forms also include the plural unless thecontext clearly dictates otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisdisclosure belongs. In the case of conflict, the present descriptionwill control.

Disorders Associated with the Spinal Cord

The spinal cord is one of two components that together characterize thecentral nervous system (CNS; brain and spinal cord). The spinal cordconnects the body to the brain, serving as a conduit for the messagesand communications necessary for movement and sensation. The spinal cordis a fragile, thin, tubular bundle made up of nerve fibers and cellbodies, as well as support cells, housed within the vertebral column.

The motor neurons and pathways of the spinal cord are important for theinitiation, execution, modification, and precision of movement. Whenthese neurons and/or pathways are damaged in some manner, such as, butnot limited to, trauma, tumorous growth, cardiovascular defects,inflammation, de-myelination, neuropathy, degeneration and/or celldeath, the consequence is typically a defect in some form of movement.Similarly, sensory neurons and pathways of the spinal cord are criticalfor proprioception and sensation, and when damaged, can result in aninability to sense certain stimuli and/or pain syndromes.

Non-limiting examples of disorders such as those described above, whichare associated with the spinal cord include, but are not limited to,motor neuron disease, amyotrophic lateral sclerosis (ALS; Lou Gehrig'sdisease), progressive bulbar palsy, pseudobulbar palsy, primary lateralsclerosis, progressive muscular atrophy, spinal muscular atrophy,post-polio syndrome, bulbar palsy, Kennedy's disease, hereditary spasticparaplegia, Friedreich's ataxia, Charcot-Marie-Tooth disease, hereditarymotor and sensory neuropahty, peroneal muscula atrophy, neuropathies,de-myelinating diseases, viral de-myelination, metabolic de-myelination,multiple sclerosis, neuromyelitis optica (Devic's disease), concentricsclerosis (Balo's sclerosis), ataxics, paraplegia, spinocerebellarataxia, acute-disseminated encephalomyelitis, complex regional painsyndrome (CPRS I and CPRS II), ataxia telangiectasia, episodic ataxia,multiple system atrophy, sporadic ataxia, lipid storage diseases,Niemann-Pick disease, Fabry disease, Faber's disease, GM1 or GM2gangliosidoses, Tay-Sachs disease, Sandhoff disease, Krabbe disease,metachromatic leukodystrophy, Machado-Joseph disease (spinocerebellarataxia type 3), meningitis, myelitis, myopathy, mitochondrial myopathy,encephalomyopathy, Barth syndrome, Chronic progressive externalophtalmoplegia, Kearns-Sayre syndrome, Leigh syndrome, mitochondrial DNAdepletion syndromes, myoclonus epilepsy with ragged red fibers, NARP(neuropathy, ataxia and retinitis pigmentosa, diseases of theneuromuscular junction, myasthenia gravis, myoclonus, neuropathic pain,neurodegenerative diseases, Parkinson's disease, Alzheimer's disease,Huntington's disease, Lewy body disease, Vitamin B12 deficiency,subacute combined degeneration of the spinal cord (Lichtheim's disease),tropical spastic paraparesis, distal hereditary motor neuronopathies,Morvan's syndrome, leukodystrophies, and/or Rett syndrome.

In one embodiment, the compositions and methods of the presentdisclosure may be used to treat any disease of the central nervoussystem.

In one embodiment, the compositions and methods of the presentdisclosure may be used to treat a disease associated with the spinalcord.

In one embodiment, the compositions and methods of the presentdisclosure may be used for the treatment of a neurodegenerative disease.

In one embodiment, the compositions and methods of the presentdisclosure may be used for the treatment of a motor neuron disease.

In one embodiment, the compositions and methods of the presentdisclosure may be used for the treatment of amyotrophic lateralsclerosis (ALS).

Amyotrophic Lateral Sclerosis (ALS) and SOD1

Amyotrophic lateral sclerosis (ALS), an adult-onset neurodegenerativedisorder, is a progressive and fatal disease characterized by theselective death of motor neurons in the motor cortex, brainstem andspinal cord. Patients diagnosed with ALS develop a progressive musclephenotype characterized by spasticity, hyperreflexia or hyporeflexia,fasciculations, muscle atrophy and paralysis. These motor impairmentsare caused by the de-innervation of muscles due to the loss of motorneurons. The major pathological features of ALS include degeneration ofthe corticospinal tracts and extensive loss of lower motor neurons(LMNs) or anterior horn cells (Ghatak et al., J Neuropathol Exp Neural.,1986, 45, 385-395), degeneration and loss of Betz cells and otherpyramidal cells in the primary motor cortex (Udaka et al., ActaNeuropathol, 1986, 70, 289-295; Maekawa et al., Brain, 2004, 127,1237-1251) and reactive gliosis in the motor cortex and spinal cord(Kawamata et al., Am J Pathol., 1992, 140,691-707; and Schiffer et al.,J Neurol Sci., 1996, 139, 27-33). ALS is usually fatal within 3 to 5years after the diagnosis due to respiratory defects and/or inflammation(Rowland L P and Shneibder N A, N Engl. Med., 2001, 344, 1688-1700).

A cellular hallmark of ALS is the presence of proteinaceous,ubiquitinated, cytoplasmic inclusions in degenerating motor neurons andsurrounding cells (e.g., astrocytes). libiquitinated inclusions (i.e.,Lewy body-like inclusions or Skein-like inclusions) are the most commonand specific type of inclusion in ALS and are found in LMNs of thespinal cord and brainstem, and in corticospinal upper motor neurons(UMNs) (Matsumoto et al., J Neural Sci., 1993, 115, 208-213; and Sasakand Maruvama, Acta Neuropathol 1994, 87, 578-585). A few proteins havebeen identified to be components of the inclusions, including ubiquitin,Cu/Zn superoxide dismutase 1 (SOD1), peripherin and Dorfin.Neurofilamentous inclusions are often found in hyaline conglomerateinclusions (HCls) and axonal ‘spheroids’ in spinal cord motor neurons inALS. Other types and less specific inclusions include Bunina bodies(cystatin C-containing inclusions) and Crescent shaped inclusions (SCIS)in upper layers of the cortex. Other neuropathological features seen inALS include fragmentation of the Golgi apparatus, mitochondrialvacuolization and ultrastructural abnormalities of synaptic terminals(Fujita et al., Acta Neuropathol, 2002, 103, 243-247).

In addition, in frontotemporal dementia ALS (FM-ALS), cortical atrophy(including the frontal and temporal lobes) is also observed, which maycause cognitive impairment in FM-ALS patients.

ALS is a complex and multifactorial disease and multiple mechanismshypothesized as responsible for ALS pathogenesis include dysfunction ofprotein degradation, glutamate excitotoxicity, mitochondrial.dysfunction, apoptosis, oxidative stress, inflammation, proteinmisfolding and aggregation, aberrant RNA metabolism, and altered geneexpression.

About 10% of ALS cases have family history of the disease, and thesepatients are referred to as familial ALS (fALS) or inherited patients,commonly with a Mendelian dominant mode of inheritance and highpenetrance. The remainder (approximately 90%-95%) is classified assporadic ALS (sALS), as they are not associated with a documented familyhistory, which is thought to be due to other risk factors, includingenvironmental factors, genetic polymorphisms, somatic mutations, andpossibly gene-environmental interactions. In most cases, familiar (orinherited) ALS is inherited as autosomal dominant disease, but pedigreeswith autosomal recessive and X-linked inheritance and incompletepenetrance exist. Sporadic and familial forms are clinicallyindistinguishable suggesting a common pathogenesis. The precise cause ofthe selective death of motor neurons in ALS remains elusive. Progress inunderstanding the genetic factors in fALS may shed light on both formsof the disease.

Recently, an explosion in research and understanding of genetic causesof ALS has led to the discovery of discovered mutations in more than 10different genes now known to cause fALS. The most common ones are foundin the genes encoding Cu/Zn superoxide dismutase 1 (SOD1: ˜20%) (Rosen DR et al., Nature, 1993, 362, 59-62), fused in sarcoma/translated inliposarcoma (FUS/ITS; 1-5%) and TDP-43 (TARDBP; 1-5%). Recently, ahexanucleotide repeat expansion (GGGGCC)_(n) in the C9orf72 gene wasidentified as the most frequent cause of fALS (˜40%) in the Westernpopulation (reviewed by Renton et al., Nat. Nenrosci., 2014, 17, 17-23).Other genes mutated in ALS include akin (ALS2), senataxin (SETX),vesicle-associated membrane protein (VAPB), angiogenin (ANG). fALS genescontrol different cellular mechanisms, suggesting that the pathogenesisof ALS is complicated and may be related to several different processesfinally leading to motor neuron degeneration.

SOD1 is one of the three human superoxide dismutases identified andcharacterized in mammals: copper-zinc superoxide dismutase (Cu/ZnSOD orSOD1), manganese superoxide dismutase (MnSOD or SOD2), and extracellularsuperoxide dismutase (ECSOD or SOD3). SOD1 is a 32 kDa homodimer of a153-residue polypeptide with one copper- and one zinc-binding site persubunit, which is encoded by SOD1 gene (GenBank access No.: NM_900454.4)on human chromosome 21 (see Table 10). SOD1 catalyzes the reaction ofsuperoxide anion (O²⁻) into molecular oxygen (O₂) and hydrogen peroxide(H₂O₂) at a hound copper ion. The intracellular concentration of SOD1 ishigh (ranging from 10 to 100 μM), accounting for 1% of the total proteincontent in the central nervous system (CNS). The protein is localizednot only in the cytoplasm but also in the nucleus, lysosomes,peroxisomes, and mitochondrial intermembrane spaces in eukaryotic cells(Lindenau J et al., Glia, 2000, 29, 25-34).

Mutations in SOD1 gene are carried by 15-20% of fALS patients and by1-2% of all ALS cases. Currently, at least 170 different mutationsdistributed throughout the 153-amino acid SOD1 polypeptide have beenfound to cause ALS, and an updated list can be found at the ALS onlineGenetic. Database (ALSOD) (Wroe R et al., Amyotroph Lateral Scier.,2008, 9, 249-250). Table 1 lists some examples of mutations in SOD1 inALS, These mutations are predominantly single amino acid substitutions(i.e. missense mutations) although deletions, insertions, and C-terminaltruncations also occur. Different SOD1 mutations display differentgeographic distribution patterns. For instance, about half of allAmericans with ALS caused by SOD1 gene mutations have a particularmutation Ala4Val (or A4V). The A4V mutation is typically associated withmore severe signs and symptoms. The 1113T mutation is by far the mostcommon mutation in the ⁻United Kingdom. The most prevalent mutation inEurope is D90A substitution.

TABLE 1 Examples of SOD1 mutations in ALS Mutations Exon1 Q22L; E21K, G;F20C; N19S; G16A, S; V14M, S; G12R; (220 bp) G10G, V, R; L8Q, V; V7E;C6G, F; V5L; A4T, V, S Exon2 T54R; E49K; H48R, Q; V47F, A; H46R; F45C;H43R; (97 bp) G41S, D; G37R; V29, insA Exon3 D76Y, V; G72S, C; L67R;P66A; N65S; S59I, S (70 bp) Exon4 D124G, V; V118L, InsAAAAC; L117V;T116T; R115G; (118 bp) G114A; I113T, F; I112M, T; G108V; L106V, F;S106L, delTCACTC; I104F; D101G, Y, H, N; E100G, K; I99V; V97L, M; D96N,V; A95T, V; G93S, V, A, C, R, D; D90V, A; A89T, V; T88delACTGCTGAC;V87A, M; N86I, S, D, K; G85R, S; L84V, F; H80R Exon5 I151T, S; I149T;V148I, G; G147D, R; C146R, stop; (461 bp) A145T, G; L144F, S; G141E,stop; A140A, G; N139D, K, H, N; G138E; T137R; S134N; E133V, delGAA,insTT; E132insTT; G127R, InsTGGG; L126S, delITT, stop; D126, delTT

To investigate the mechanism of neuronal death associated with SOD1 genedefects, several rodent models of SOD1-linked ALS were developed in theart, which express the human SOD1 gene with different mutations,including missense mutations, small deletions or insertions. Someexamples of ALS mouse models include SOD1^(G93A), SOD1^(A4V),SOD1^(G37R), SOD1^(G85R), SOD1^(D90A), SOD1^(L84V), SOD1^(1113T),SOD1^(H36R/H48Q), SOD1^(G127X), SOD1^(L126X) and SOD1^(L126delTT). Thereare two transgene rat models carrying two different human SOD1mutations: SOD1^(H46R) and SOD1^(G93R). These rodent ALS models candevelop muscle weakness similar to human ALS patients and otherpathogenic features that reflect several characteristics of the humandisease, in particular, the selective death of spinal motor neurons,aggregation of protein inclusions in motor neurons and microglialactivation. It is well known in the art that the transgenic rodents aregood models of human SOD1-assocaited ALS disease and provide models forstudying disease pathogenesis and developing disease treatment.

Studies in animal and cellular models showed that SOD1 pathogenicvariants cause ALS by gain of function. That is to say, the superoxidedismutase enzyme gains new but harmful properties when altered by SOD1mutations. For example, some SOD1 mutated variants in ALS increaseoxidative stress (e.g., increased accumulation of toxic superoxideradicals) by disrupting redox cycle. Other studies also indicate thatsome SOD1 mutated variants in ALS might acquire toxic properties thatare independent of its normal physiological function (such as abnormalaggregation of tnisfolded SOD1 variants). In the aberrant redoxchemistry model, mutant SOD1 is unstable and through aberrant chemistryinteracts with nonconventional substrates causing reactive oxygenspecies (ROS) overproduction. In the protein toxicity model, unstable,misfolded SOD1 aggregates into cytoplasmic inclusion bodies,sequestering proteins crucial for cellular processes. These twohypotheses are not mutually exclusive. It has been shown that oxidationof selected histidine residues that bind metals in the active sitemediates SOD1 aggregation.

The aggregated mutant SOD 1 protein may also induce mitochondrialdysfunction (Vehvilainen Petal., Front Cell Neztrosci., 2014, 8, 126),impairment of axonal transport, aberrant RNA metabolism, glial cellpathology and glutamate excitotoxicity. In some sporadic ALS cases,misfolded wild-type SOD1 protein is found in diseased motor neuronswhich forms “toxic conformation” that is similar to familial ALS-linkedSOD1 variants (Rotunno M S and Bosco D A, Front Cell Neurosci., 2013,16, 7, 253). Such evidence suggests that ALS is a protein misfoldingdisease analogous to other neurodegenerative diseases such asAlzheimer's disease and Parkinson's disease.

Currently, no curative treatments are available for patients sufferingfrom ALS. Until recently, the only FDA approved drug was Riluzole (alsocalled. RILUTEK®), an inhibitor of glutamate release, with a moderateeffect on ALS, only extending survival by 2-3 months if it is taken for18 months. Unfortunately, patients taking riluzole do not experience anyslowing in disease progression or improvement in muscle function.Therefore, riluzole does not present a cure, or even an effectivetreatment. In 2017, the FDA approved Radicava (edaravone) for thetreatment of ALS, the first such approval in 22 years. Administeredintravenously and serving as a free-radical scavenger and anti-oxidant,Radicava has been shown to slow disease progression. In a clinical Phase3 trial (NCT01492686) of 137 patients, Radicava slowed the decline inphysical function as compared to those patients taking placebo and asdetermined by score on the ALS Functional Rating Scale-Revised(ALSFRS-R) (Writing group; Edaravone (MCI-186) ALS 19 Study Group LancetNeural. 2017 July; 16(7):505-512). The approval of Radicava isconsidered an advance in terms of treatment of ALS, however it is stillnot a cure. Researchers continue to search for better therapeuticagents.

One approach to inhibit abnormal SOD1 protein aggregation is tosilence/inhibit SOD1 gene expression in ALS. It has been reported thatsmall interfering RNAs for specific gene silencing of the mutated alleleis therapeutically beneficial for the treatment of fALS (e.g., Raigh G Set al., Nat. Medicine, 2005, 11(4), 429-433; and Raoul C et al., Nat.Medicine, 2005, 11(4), 423-428; and Maxwell M M et al., PNAS, 2004,101(9), 3178-3183; and Ding H et al., Chinese Medical J., 2011, 124(1),106-110; and Scharz D S et al., Plos Genet., 2006, 2(9), e140; thecontent of each of which is incorporated herein by reference in theirentirety).

Many other RNA therapeutic agents that target SOD1 gene and modulateSOD1 expression in ALS are taught in the art, such RNA based agentsinclude antisense oligonucleotides and double stranded small interferingRNAs. See, e.g., Wang H et al., J Biol. Chem 2008, 283(23),15845-15852); U.S. Pat. Nos. 7,498,316; 7,632,938; 7,678,895; 7,951,784;7,977,314; 8,183,219; 8,309,533 and 8, 586, 554; and U.S. Patentpublication Nos. 2006/0229268 and 2011/0263680; the content of each ofwhich is herein incorporated by reference in their entirety.

The present disclosure employs viral vectors such as adeno-associatedviral (AAV) vectors to deliver siRNA duplexes or SOD1 targetingpolynucleotides into cells with high efficiency. The AAV vectorscomprising RNAi molecules, e.g., siRNA molecules of the presentdisclosure may increase the delivery of active agents into motorneurons. SOD1 targeting polynucleotides may be able to inhibit SOD1.gene expression (e.g., mRNA level) significantly inside cells;therefore, ameliorating SOD1 expression induced stress inside the cellssuch as aggregation of protein and formation of inclusions, increasedfree radicals, mitochondrial dysfunction and RNA metabolism.

Such SOD1 targeting polynucleotides may be used for treating ALS.According to the present disclosure, methods thr treating and/orameliorating ALS in a patient comprises administering to the patient aneffective amount of at least one SOD1 targeting polynucleotide encodingone or more siRNA duplexes into cells and allowing theinhibition/silence of SOD1 gene expression, are provided.

Compositions Vectors

In some embodiments, the siRNA molecules described herein can beinserted into, or encoded by, vectors such as plasmids or viral vectors.Preferably, the siRNA molecules are inserted into, or encoded by, viralvectors.

Viral vectors may be Herpesvirus (HSV) vectors, retroviral vectors,adenoviral vectors, adeno-associated viral vectors, lentiviral vectors,and the like. In some specific embodiments, the viral vectors are AAVvectors.

Retroviral Vectors

In some embodiments, the siRNA duplex targeting SOD1 gene may be encodedby a retroviral vector (See, e.g., U.S. Pat. Nos. 5,399,346; 5,124,263;4,650,764 and 4,980,289; the content of each of which is incorporatedherein by reference in their entirety).

Adenoviral Vectors

Adenoviruses are eukaryotic DNA viruses that can be modified toefficiently deliver a nucleic acid to a variety of cell types in vivo,and have been used extensively in gene therapy protocols, including fortargeting genes to neural cells. Various replication defectiveadenovirus and minimum adenovirus vectors have been described fornucleic acid therapeutics (See, e.g., PCT Patent Publication Nos.WO199426914, WO 199502697, WO199428152, WO199412649, WO199502697 andWO199622378; the content of each of which is incorporated by referencein their entirety). Such adenoviral vectors may also be used to deliversiRNA molecules of the present disclosure to cells.

Adeno-Associated Viral (AAV) Vectors

An AAV is a dependent parvovirus. Like other parvovinises, AAV is asingle stranded, non-enveloped DNA virus, having a genome of about 5000nucleotides in length containing two open reading frames that encode theproteins responsible for replication (Rep) and the structural protein ofthe capsid (Cap). The open reading frames are flanked by two InvertedTerminal Repeat (ITR) sequences, which serve as the origin ofreplication of viral genome. Furthermore, the AAV genome contains apackaging sequence, allowing packaging of the viral genome into an AAVcapsid. The AAV vector requires co-helper (e.g., adenovims) to undergo aproductive infection in infected cells. in the absence of such helperfunctions, the AAV virions essentially enter host cells and integrateinto cells genome.

AAV vectors have been investigated for siRNA delivery because of itsseveral unique features. These features include (i) ability to infectboth dividing and non-dividing cells; (ii) a broad host range forinfectivity, including human cells; (iii) wild-type AAV has never beenassociated with any disease and cannot replicate in infected cells; (iv)lack of cell-mediated immune response against the vector and (v) abilityto integrate into a host chromosome or persist episomally, therebycreating potential for long-term expression. Moreover, infection withAAV vectors has minimal influence on changing the pattern of cellulargene expression (Stilwell and Samulski et al., Biotechniques, 2003, 34,148).

Typically. AAV vectors for siRNA delivery may be recombinant viralvectors which are replication defective because of lacking sequencesencoding functional Rep and Cap proteins in viral genome. In some cases,the defective AAV vectors may lack most of all coding sequences andessentially only contains one or two AAV ITR sequences and a packagingsequence.

AAV vectors may also comprise self-complementary AAV vectors (scAAVs).scAAV vectors contain both DNA strands which anneal together to formdouble stranded DNA. By skipping second strand synthesis, scAAVs allowfor rapid expression in the cell.

Methods for producing/modifying AAV vectors arc disclosed in the artsuch as pseudotyped AAV vectors (PCT Patent Publication Nos.WO200028004; WO200123001; WO2004112727; WO 2005005610 and WO 2005072364,the content of each of which is incorporated herein by reference intheir entirety).

AAV vectors for delivering siRNA molecules into mammalian cells, may beprepared or derived from various serotypes of AAVs, including, but notlimited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ8 andAAV-DJ. In some cases, different serotypes of AAVs may be mixed togetheror with other types of viruses to produce chimeric AAV vectors.

In one embodiment, the AAV serotype is AAVrh10.

AAV vectors for siRNA delivery may be modified to enhance the efficiencyof delivery. Such modified AAV vectors containing the siRNA expressioncassette can be packaged efficiently and can be used to infectsuccessfully the target cells at high frequency and with minimaltoxicity.

In some embodiments, the AAV vector for delivering siRNA duplexes of thepresent disclosure may be a human serotype AAV vector. Such human AAVvector may be derived from any known serotype, e.g., from any one ofserotypes AAV1-AAV11. As non-limiting examples, AAV vectors may bevectors comprising an AAV1-derived genome in an AAV1-derived capsid;vectors comprising an AAV2-derived genome in an AAV2-derived genome;vectors comprising an AAV4-derived genome in an AAV4 derived capsid;vectors comprising an AAV6-derived genome in an AAV6 derived capsid orvectors comprising an AAV9-derived genome in an AAV9 derived capsid.

In other embodiments, the AAV vector for delivering siRNA duplexes ofthe present disclosure may be a pseudotyped AAV vector which containssequences and/or components originating from at least two different AAVserotypes. Pseudotyped AAV vectors may be vectors comprising an AAVgenome derived from one AAV serotype and a Capsid protein derived atleast in part from a different AAV serotype. As non-limiting examples,such pseudotyped AAV vectors may be vectors comprising an AAV2-derived.genome in an AAV1-derived capsid; or vectors comprising an AAV2-derivedgenome in an AAV6-derived capsid; or vectors comprising an AAV2-derivedgenome in an AAV4-derived capsid; or an AAV2-derived genome in anAAV9-derived capsid.

In other embodiments, AAV vectors may be used for delivering siRNAmolecules to the central nervous system (e.g., U.S. Pat. No. 6,180,613;the content of which is herein incorporated by reference in itsentirety).

In some aspects, the AAV vector for delivering siRNA duplexes of thepresent disclosure may further comprise a modified capsid includingpeptides from non-viral origin. In other aspects, the AAV vector maycontain a CNS specific chimeric capsid to facilitate the delivery ofsiRNA. duplexes into the brain and the spinal cord. For example, analignment of cap nucleotide sequences from AAV variants exhibiting CNStropism may be constructed to identify variable region (VR) sequence andstructure.

The present disclosure refers to structural capsid proteins (includingVP1, VP2 and VP3) which are encoded by capsid (Cap) genes. These capsidproteins form an outer protein structural shell (i.e. capsid) of a viralvector such as AAV. VP capsid proteins synthesized from Cappolynucleotides generally include a methionine as the first amino acidin the peptide sequence (Met1), which is associated with the start codon(AUG or ATG) in the corresponding Cap nucleotide sequence. However, itis common for a first-methionine (Met1) residue or generally any firstamino acid (AA1) to be cleaved off after or during polypeptide synthesisby protein processing enzymes such as Met-aminopeptidases. This“Met/AA-clipping” process often correlates with a correspondingacetylation of the second amino acid in the polypeptide sequence (e.g.,alanine, valine, serine, threonine, etc.). Met-clipping commonly occurswith VP1 and VP3 capsid proteins but can also occur with VP2 capsidproteins.

Where the Met/AA-clipping is incomplete, a mixture of one or more (one,two or three) VP capsid proteins comprising the viral capsid may beproduced, some of which may include a Met1/AA1 amino acid (Met+/AA+) andsome of which may lack a Met1/AA1 amino acid as a result ofMet/AA-clipping (Met−/AA−). For further discussion regardingMet/AA-clipping in capsid proteins, see fin, et al. Direct LiquidChromatography/Mass Spectrometry Analysis for Complete Characterizationof Recombinant Adeno-Associated Virus Capsid Proteins. Hum Gene TherMethods. 2017 Oct. 28(5):255-267; Hwang, et al. N-Terminal Acetylationof Cellular Proteins Creates Specific Degradation Signals. Science. 2010Feb. 19. 327(5968): 973-977; the contents of which are each incorporatedherein by reference in its entirety.

According to the present disclosure, references to capsid proteins isnot limited to either clipped (Met−/AA−) or unclipped (Met+/AA+) andmay, in context, refer to independent capsid proteins, viral capsidscomprised of a mixture of capsid proteins, and/or polynucleotidesequences (or fraaments thereof) which encode, describe, produce orresult in capsid proteins of the present disclosure. A direct referenceto a “capsid protein” or “capsid polypeptide” (such as VP1, VP2 or VP2)may also comprise VP capsid proteins which include a Met1/AA1 amino acid(Met+/AA+) as well as corresponding VP capsid proteins which lack theMet1/AA1 amino acid as a result of Met/AA-clipping (Met−/AA−).

Further according to the present disclosure, a reference to a specificSEQ ID NO: (whether a protein or nucleic acid) which comprises orencodes, respectively, one or more capsid proteins which include aMet1/AA1 amino acid (Met+/AA+) should be understood to teach the VPcapsid proteins which lack the Met1/AA1 amino acid as upon review of thesequence, it is readily apparent any sequence which merely lacks thefirst listed amino acid (whether or not Met1/AA1).

As a non-limiting example, reference to a VP1 polypeptide sequence whichis 736 amino acids in length and which includes a “Met1” amino acid(Met+) encoded by the AUG/ATG start codon may also be understood toteach a VP1 polypeptide sequence which is 735 amino acids in length andwhich does not include the “Met1” amino acid (Met−) of the 736 aminoacid Met+ sequence. As a second non-limiting example, reference to a VP1polypeptide sequence which is 736 amino acids in length and whichincludes an “AA1” amino acid (AA1+) encoded by any NNN initiator codonmay also be understood to teach a VP1 polypeptide sequence which is 735amino acids in length and which does not include the “AA1” amino acid(AA1−) of the 736 amino acid AA1+ sequence.

References to viral capsids formed from VP capsid proteins (such asreference to specific AAV capsid serotypes), can incorporate VP capsidproteins which include a Met1/AA1 amino acid (Met+/AA 1+), corresponding\/P capsid proteins which lack the Met1/AA1 amino acid as a result ofMet/AA1-clipping (Met−/AA1−), and combinations thereof (Met+/AA1+ andMet−/AA1−).

As a non-limiting example, an AAV capsid serotype can include VP1(Met+/AA1+), VP1 (Met−/AA1−), or a combination of VP1 (Met+/AA1+) andVP1 (Met-/AA1−). An AAV capsid serotype can also include VP3(Met+/AA1+), VP3 (Met−/AA1−) or a combination of VP3 (Met+/AA1+) and VP3(Met−/AA1−); and can also include similar optional combinations of VP2(Met+/AA1) and VP2 (Met−/AA1−).

Viral Genome

In one embodiment, as shown in an AAV particle comprises a viral genomewith a payload region.

Viral Genome Size

In one embodiment, the viral genome which comprises a payload describedherein, may be single stranded or double stranded viral genome. The sizeof the viral genome may be small, medium, large or the maximum size.Additionally, the viral genome may comprise a promoter and a polyA tail.

In one embodiment, the viral genome which comprises a payload describedherein, may be a small single stranded viral genome. A small singlestranded viral genome may be 2.7 to 3.5 kb in size such as about 2.7,2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, and 3.5 kb in size. As a non-limitingexample, the small single stranded viral genome may be 3.2 kb in size.Additionally, the viral genome may comprise a promoter and a polyA tail.

In one embodiment, the viral genome which comprises a payload describedherein, may be a small double stranded viral genome. A small doublestranded viral genome may be 1.3 to 1..7 kb in size such as about 1.3,1.4, 1.5, 1.6, and 1.7 kb in size. As a non-limiting example, the smalldouble stranded viral genome may be 1.6 kb in size. Additionally, theviral genome may comprise a promoter and a polyA tail.

In one embodiment, the viral genome which comprises a payload describedherein, may a medium single stranded viral genome. A medium singlestranded viral genome may be 3.6 to 4.3 kb in size such as about 3.6,3.7, 3.8, 3.9, 4.0, 4.1, 4.2 and 4.3 kb in size. As a non-limitingexample, the medium single stranded viral genome may be 4.0 kb in size.Additionally, the viral genome may comprise a promoter and a polyA tail.

In one embodiment, the viral genome which comprises a payload describedherein, may be a medium double stranded viral genome. A medium doublestranded viral genome may be 1.8 to 2.1 kb in size such as about 1.8,1.9, 2.0, and 2.1 kb in size. As a non-limiting example, the mediumdouble stranded viral genome may be 2.0 kb in size. Additionally, theviral genome may comprise a promoter and a polyA tail.

In one embodiment, the viral genome which comprises a payload describedherein, may be a large single stranded viral genome. A large singlestranded viral genome may be 4.4 to 6.0 kb in size such as about 4.4,4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,5.9 and 6.0 kb in size. As a non-limiting example, the large singlestranded viral genome may be 4.7 kb in size. As another non-limitingexample, the large single stranded viral genome may be 4.8 kb in size.As yet another non-limiting example, the large single stranded viralgenome may be 6.0 kb in size. Additionally, the viral genome maycomprise a promoter and a polyA tail.

In one embodiment, the viral genome which comprises a payload describedherein, may be a large double stranded viral genome. A large doublestranded viral genome may be 2.2 to 3.0 kb in size such as about 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 and 3.0 kb in size. As a non-limitingexample, the large double stranded viral genome may be 2.4 kb in size.Additionally, the viral genome may comprise a promoter and a polyA tail.

Viral Genome Component: Inverted Terminal Repeats (ITRs)

The AAV particles of the present disclosure comprise a viral genome withat least one ITR region and a payload region. In one embodiment theviral genome has two ITRs. These two ITRs flank the payload region atthe 5′ and 3′ ends. The ITRs function as origins of replicationcomprising recognition sites for replication. ITRs comprise sequenceregions which can be complementary and symmetrically arranged. ITRsincorporated into viral genomes of the disclosure may be comprised ofnaturally occurring polynucleotide sequences or recombinantly derivedpolynucleotide sequences.

The ITRs may be derived from the same serotype as the capsid, selectedfrom any of the serotypes herein, or a derivative thereof. The ITR maybe of a different serotype from the capsid. In one embodiment the AAVparticle has more than one ITR. In a non-limiting example, the AAVparticle has a viral genome comprising two ITRs. In one embodiment theITRs are of the same serotype as one another. In another embodiment theITRs are of different serotypes. Non-limiting examples include zero, oneor both of the ITRs having the same serotype as the capsid. In oneembodiment both ITRs of the viral genome of the AAV particle are AAV2ITRs.

Independently, each ITR may be about 100 to about 150 nucleotides inlength. An ITR may be about 100-105 nucleotides in length, 106-110nucleotides in length, 111-115 nucleotides in length, 116-120nucleotides in length. 121-125 nucleotides in length, 126-130nucleotides in length, 131-135 nucleotides in length, 136-140nucleotides in length, 141-145 nucleotides in length or 146-150nucleotides in length. In one embodiment the ITRs are 140-142nucleotides in length. Non-limiting examples of ITR length are 102, 140,141, 142, 145 nucleotides in length, and those having at least 95%identity thereto.

In one embodiment, the AAV particle comprises a nucleic acid sequenceencoding an siRNA molecule which may be located near the 5′ end of theflip ITR in an expression vector. In another embodiment, the AAVparticle comprises a nucleic acid sequence encoding an siRNA moleculemay be located near the 3′ end of the flip ITR in an expression vector.In yet another embodiment, the AAV particle comprises a nucleic acidsequence encoding an siRNA molecule may be located near the 5′ end ofthe flop ITR in an expression vector. In yet another embodiment, the AAVparticle comprises a nucleic acid sequence encoding an siRNA moleculemay be located near the 3′ end of the flop ITR in an expression vector.In one embodiment, the AAV particle comprises a nucleic acid sequenceencoding an siRNA molecule may be located between the 5′ end of the flipITR and the 3′ end of the flop ITR in an expression vector. In oneembodiment, the AAV particle comprises a nucleic acid sequence encodingan siRNA molecule may be located between (e.g., half-way between the 5′end of the flip ITR and 3′ end of the flop ITR or the 3′ end of the flopITR and the 5′ end of the flip ITR), the 3′ end of the flip ITR and the5′ end of the flip ITR in an expression vector. As a non-limiting,example, the AAV particle comprises a nucleic acid sequence encoding ansiRNA molecule may be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30 or more than 30 nucleotides downstream from the 5′ or 3′ end of anITR (e.g., Flip or Flop ITR) in an expression vector. As a non-limitingexample, the AAV particle comprises a nucleic acid sequence encoding ansiRNA molecule may be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29,30 or more than 30 nucleotides upstream from the 5′ or 3′ end of an ITR(e.g., Flip or Flop ITR) in an expression vector. As anothernon-limiting example. the AAV particle comprises a nucleic acid sequenceencoding an siRNA molecule may be located within 1-5, 1-10, 1-15, 1-20,1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30,15-20, 15-25, 15-30, 20-25, 20-30 or 25-30 nucleotides downstream fromthe 5′ or 3′ end of an FIR (e.g., Flip or Flop ITR) in an expressionvector. As another non-limiting example, the AAV particle comprises anucleic acid sequence encoding an siRNA molecule may be located within1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15,10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30 or 25-30 upstreamfrom the 5′ or 3′ end of an ITR (e.g., Flip or Flop ITR) in anexpression vector, As a non-limiting example, the AAV particle comprisesa nucleic acid sequence encoding an siRNA molecule may be located withinthe first 1©′0 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% ormore than 25% of the nucleotides upstream from the 5′ or 3′ end of anITR (e.g., Flip or Flop ITR) in an expression vector. As anothernon-limiting example, the AAV particle comprises a nucleic acid sequenceencoding an siRNA molecule may be located with the first 1-5%, 1-10%,1-15%, 1-20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%, 10-15%, 10-20%, 10-25%,15-20%, 15-25%, or 20-25% downstream from the 5′ or 3′ end of an ITR(e.g., Flip or Flop ITR) in an expression vector.

Viral Genome Component: Promoters

In one embodiment, the payload region of the viral genome comprises atleast one element to enhance the transgene target specificity andexpression (See e.g., Powell et al. Viral Expression Cassette Elementsto Enhance Transgene Target Specificity and Expression in Gene Therapy,2015; the contents of which are herein incorporated by reference in itsentirety). Non-limiting examples of elements to enhance the transgenetarget specificity and expression include promoters, endogenous miRNAs,post-transcriptional regulatory elements (PREs), polyadenylation (PolyA)signal sequences and upstream enhancers (USEs), CMV enhancers andintrons.

A person skilled in the art may recognize that expression of thepolypeptides of the disclosure in a target cell may require a specificpromoter, including but not limited to, a promoter that is speciesspecific, inducible, tissue-specific, or cell cycle-specific (Parr etal., Nat. Med. 3:1145-9 (1997); the contents of which are hereinincorporated by reference in their entirety).

In one embodiment, the promoter is deemed to be efficient when it drivesexpression of the polypeptide(s) encoded in the payload region of theviral genome of the AAV particle.

In one embodiment, the promoter is a promoter deemed to be efficient todrive the expression of the modulatory polynucleotide.

In one embodiment, the promoter is a promoter deemed to be efficientwhen it drives expression in the cell being targeted.

In one embodiment, the promoter drives expression of the payload for aperiod of time in targeted tissues. Expression driven by a promoter maybe for a period of 1 hour, 2, hours, 3 hours, 4 hours, 5 hours, 6 hours,7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6months, 7 months, 8 months, 9 months, 10 months. 11 months, 1 year, 13months, 14 months, 15 months, 16 months, 17 months, 18 months, 19months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or morethan 10 years. Expression may be for 1-5 hours, 1-12 hours, 1-2 days,1-5 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months, 1-4 months, 1-6months, 2-6 months, 3-6 months, 3-9 months, 4-8 months, 6-12 months, 1-2years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8 6 years or 5-10years.

In one embodiment, the promoter drives expression of the payload for atleast 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3years 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18years, 19 years, 20 years, 21 years, 22 years, 23 years. 24 years. 25years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years, 32years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46years, 47 years, 48 years, 49 years, 50 years, 55 years, 60 years, 65years, or more than 65 years.

Promoters may be naturally occurring or non-naturally occurring.Non-limiting examples of promoters include viral promoters, plantpromoters and mammalian promoters. In some embodiments, the promotersmay be human promoters. In some embodiments, the promoter may betruncated.

Promoters which drive or promote expression in most tissues include, butare not limited to, human elongation factor 1α-subunit (EF1α),cytomegalovirus (CMV) immediate-early enhancer and/or promoter, chickenβ-actin (CBA) and its derivative CAG, glucuronidase (GUSB), or ubiquitinC (LTC). Tissue-specific expression elements can be used to restrictexpression to certain cell types such as, but not limited to, musclespecific promoters, B cell promoters, monocyte promoters, leukocytepromoters, macrophage promoters, pancreatic acinar cell promoters,endothelial cell promoters, lung tissue promoters, astrocyte promoters,or nervous system promoters which can be used to restrict expression toneurons, astrocytes, or oligodendrocytes.

Non-limiting examples of muscle-specific promoters include mammalianmuscle creatine kinase (MCK) promoter, mammalian desmin (DES) promoter,mammalian troponin I (TNNI2) promoter, and mammalian skeletalalpha-actin (ASKA) promoter (see, e.g. U.S. Patent Publication US20110212529, the contents of which are herein incorporated by referencein their entirety)

Non-limiting examples of tissue-specific expression elements for neuronsinclude neuron-specific enolase (NSE), platelet-derived growth factor(PDGF), platelet-derived growth factor B-chain (PDGF-β), synapsin (Syn),methyl-CpG binding protein 2 (MeCP2), Ca²⁺/calmodulin-dependent proteinkinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2),neurofilament light (NFL) or heavy (NFH), β-globin minigene nβ2,preproenkephalin (PPE), enkephalin (Enk) and excitatory amino acidtransporter 2 (EAAT2) promoters. Non-limiting examples oftissue-specific expression elements for astrocytes include glialfibrillary acidic protein (GFAP) and EAAT2 promoters. A non-limitingexample of a tissue-specific expression element for oligodendrocytesincludes the myelin basic protein (MBP) promoter.

In one embodiment, the promoter may be less than 1 kb. The promoter mayhave a length of 200, 210. 220, 230, 240, 250, 260, 270, 280. 290, 300,310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440,450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580,590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720,730, 740, 750, 760, 770, 780, 790, 800 or more than 800 nucleotides. Thepromoter may have a length between 200-300, 200-400, 200-500, 200-600,200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500,400-600, 400-700, 400-800 500-600 500-700, 500-800, 600-700, 600-800 or700-800.

In one embodiment, the promoter may be a combination of two or morecomponents of the same or different stalling or parental promoters suchas, but not limited to, CMV and CBA. Each component may have a length of200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,340, 350, 360, 370, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389.390, 400. 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520,530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660,670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 ormore than 800. Each component may have a length between 200-300,200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600,300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700,500-800, 600-700, 600-800 or 700-800. In one embodiment, the promoter isa combination of a 382 nucleotide CMV-enhancer sequence and a 260nucleotide CBA-promoter sequence.

In one embodiment, the viral genome comprises a ubiquitous promoter.Non-limiting examples of ubiquitous promoters include CMV, CBA(including derivatives CAG, CBh, etc.), EF-1α, PGK, UBC, GUSB (hGBp),and UCOE (promoter of HNRPA2B1-CBX3).

Yu et al. (Molecular Pain 2011, 7:63; the contents of which are hereinincorporated by reference in their entirety) evaluated the expression ofeGFP under the CAG, EF1α, PGK and. UBC promoters in rat DRG cells andprimary DRG cells using lentiviral vectors and found that UBC showedweaker expression than the other 3 promoters and only 10-12% glialexpression was seen for all promoters. Soderbloin et al. (E. Neuro 2015;the contents of ch are herein incorporated by reference in its entirety)evaluated the expression of eGFP in AAV8 with CMV and. UBC promoters andAAV2 with the CMV promoter after injection in the motor cortex.Intranasal administration of a plasmid containing a UBC or EFIα promotershowed a sustained airway expression greater than the expression withthe CMV promoter (See e.g., Gill et at., Gene Therapy 2001, Vol. 8,1539-1546; the contents of which are herein incorporated by reference intheir entirety). Husain et al. (Gene Therapy 2009; the contents of whichare herein incorporated by reference in its entirety) evaluated an HβHconstruct with a hGUSB promoter, an HSV-ILAT promoter and an NSE:promoter and found that the HβH construct showed weaker expression thanNSE in mouse brain. Passini and Wolfe (J. Virol. 2001, 12382-12392, thecontents of which are herein incorporated by reference in its entirety)evaluated the long-term effects of the HβH vector following anintraventricular injection in neonatal mice and found that there wassustained expression for at least 1 year. Low expression in all brainregions was found by Xu et al. (Gene Therapy 2001, 8, 1323-1332; thecontents of which are herein incorporated by reference in theirentirety) when NFL and NFH promoters were used as compared to theCMV-lacZ, CMV-luc, EF, GFAP, hENK, nAChR, PPE, PPE+wpre, NSE (0.3 kb),NSE (1.8 kb) and NSE (1.8 kb+wpre). Xu. et al. found that the promoteractivity in descending order was NSF, (1.8 kb), EF, NSE (0.3 kb), GRAP,CMV, hENK, PPE. NFL and NFH. NFL is a 650 nucleotide promoter and NFH isa 920 nucleotide promoter which are both absent in the liver but NFH isabundant in the sensory proprioceptive neurons, brain and spinal cordand NFH is present in the heart. Scn8a is a 470 nucleotide promoterwhich expresses throughout the DRG, spinal cord and brain withparticularly high expression seen in the hippocampal neurons andcerebellar Purkinje cells, cortex, thalamus and hypothalamus (See Drewset al. Identification of evolutionary conserved, functional noncodingelements in the promoter region of the sodium channel gene SCN8A, MammGenome (2007) 18:723-731; and Raymond et al. Expression of AlternativelySpliced Sodium Channel α-subunit genes, Journal of Biological Chemistry(2004) 279(44) 46234-46241; the contents of each of which are hereinincorporated by reference in their entireties).

Any of promoters taught by the aforementioned Yu, Soderblom, Gill,Husain, Passini, Xu, Drews or Raymond may be used in the presentdisclosure.

In one embodiment, the promoter is not cell specific.

In one embodiment, the promoter is a ubiquitin c (UBC) promoter. The UBCpromoter may have a size of 300-350 nucleotides. As a non-limitingexample, the UBC promoter is 332 nucleotides,

In one embodiment, the promoter is a β-glucuronidase (GUSB) promoter,The GUSB promoter may have a size of 350-400 nucleotides. As anon-limiting example, the GUSB promoter is 378 nucleotides.

In one embodiment, the promoter is a neurofilament light (NFL) promoter,The NFL promoter may have a size of 600-700 nucleotides. As anon-limiting example, the NFL promoter is 650 nucleotides. As anon-limiting example, the construct may be AAV-promoter-CMV/globinintron-modulatory polynucleotide-RBG, where the AAV may beself-complementary and the AAV may be the DJ serotype.

In one embodiment, the promoter is a neurofilament heavy (NFU) promoter.The NFH promoter may have a size of 900-950 nucleotides. As anon-limiting example, the NFH promoter is 920 nucleotides, As anon-limiting example, the construct may be AAV-promoter-CMV/globinintron-modulatory polynucleotide-RBG, where the AAV may beself-complementary and the AAV may be the DJ serotype.

In one embodiment, the promoter is a scn8a promoter. The scn8a promotermay have a size of 450-500 nucleotides. As a non-limiting example, thescn8a promoter is 470 nucleotides. As a non-limiting example, theconstruct may be AAV-promoter-CMV/globin intron-modulatorypolynucleotide-RBG, where the AAV may be self-complementary and the AAVmay be the DJ serotype

In one embodiment, the viral genome comprises a Pol III promoter.

In one embodiment, the viral genome comprises a P1 promoter.

In one embodiment, the viral genome comprises an FXN promoter.

In one embodiment, the promoter is a phosphoglycerate kinase I (PGK)promoter.

In one embodiment, the promoter is a chicken β-actin (CBA) promoter.

In one embodiment, the promoter is a CAG promoter which is a constructcomprising the cytomegalovirus (CMV) enhancer fused to the chickenbeta-actin (CBA) promoter.

In one embodiment, the promoter is a cytomegalovirus (CMV) promoter.

In one embodiment, the viral genome comprises a H1 promoter.

In one embodiment, the viral genome comprises a U6 promoter.

In one embodiment, the promoter is a liver or a skeletal musclepromoter. Non-limiting examples of liver promoters include humancm-1-antitrypsin (hAAT) and thyroxine binding globulin (TBG).Non-limiting examples of skeletal muscle promoters include Desmnin, NICKor synthetic C5-12.

In one embodiment, the promoter is an RNA pol III promoter. As anon-limiting example, the RNA pol III promoter is U6. As a non-limitingexample, the RNA pol III promoter is H1.

In one embodiment, the viral genome comprises two promoters. As anon-limiting example, the promoters are an EF1α promoter and a CMVpromoter.

In one embodiment, the viral genome comprises an enhancer element, apromoter and/or a 5′UTR intron. The enhancer element, also referred toherein as an “enhancer,” may be, but is not limited to, a CMV enhancer,the promoter may be, but is not limited to, a CMV, CBA, UBC, GUSB, NSE,Synapsin, MeCP2, and GFAP promoter and the 5′UTR/intron may be, but isnot limited to SV40, and CBA-MVM. As a non-limiting example, theenhancer, promoter and/or intron used in combination may be: (1) CMVenhancer, CMV promoter, SV40 5′UTR intron; (2) CMV enhancer, CBApromoter, SV 40 5′UTR intron; (3) CMV enhancer, CBA promoter, CBA-MVM5′UTR. intron; (4) UBC promoter; (5) GUSB promoter; (6) NSE promoter;(7) Synapsin promoter; (8) MeCP2 promoter, (9) GFAP promoter, (10) H1promoter; and (11) U6 promoter.

In one embodiment, the viral genome comprises an engineered promoter.

In another embodiment the viral genome comprises a promoter from anaturally expressed protein.

Viral Genome Component: Untranslated Regions (UTRs)

By definition, wild type untranslated regions (UTRs) of a gene aretranscribed but not translated. Generally, the 5′ UTR starts at thetranscription start site and ends at the start codon and the 3′ UTRstarts immediately following the stop codon and continues until thetemunation signal for transcription.

Features typically found in abundantly expressed genes of specifictarget organs may be engineered into UTRs to enhance the stability andprotein production. As a non-limiting example, a 5′ UTR from mRNAnominally expressed in the liver (e.g., albumin, serum amyloid A,Apolipoprotein AIB/E, transferrin, alpha fetoprotein, erythropoietin, orFactor VIII) may be used in the viral genomes of the AAV particles ofthe disclosure to enhance expression in hepatic cell lines or liver.

While not wishing to be bound by theory, wild-type 5′ untranslatedregions (UTRs) include features which play roles in translationinitiation. Kozak sequences, which are commonly known to be involved inthe process by which the ribosome initiates translation of many genes,are usually included in 5′ UTRs. Kozak sequences have the consensusCCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three basesupstream of the start codon (ATG), which is followed by another ‘G’.

In one embodiment, the 5′UTR in the viral genome includes a Kozaksequence.

In one embodiment, the 5′UTR in the viral genome does not include aKozak sequence.

While not wishing to he bound by theory, wild-type 3′ UTRs are known tohave stretches of Adenosines and Uridines embedded therein. These AUrich signatures are particularly prevalent in aenes with high rates ofturnover. Based on their sequence features and functional properties,the AU rich elements (AREs) can be separated into three classes (Chen etal, 1995, the contents of which are herein incorporated by reference inits entirety): Class I ARES, such as, but not limited to, c-Myc andMyoD, contain several dispersed copies of an AUUUA motif within U-richregions. Class II AREs, such as, but not limited to, GM-CSF and TNF-a,possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Class IIIARES, such as, but not limited to, c-Jun and Myogenin, are less welldefined. These U rich regions do not contain an AUUUA motif. Mostproteins binding to the AREs are known to destabilize the messenger,whereas members of the HAV family, most notably HuR, have beendocumented to increase the stability of mRNA. HuR binds to AREs of allthe three classes. Engineering the HuR specific binding sites into the3′ UTR of nucleic acid molecules will lead to HuR binding and thus,stabilization of the message in vivo.

Introduction, removal or modification of 3′ UTR AU rich elements (AREs)can be used to modulate the stability of polynucleotides. Whenengineering specific polynucleotides, e.g., payload regions of viralgenomes, one or more copies of an ARE can be introduced to makepolynucleotides less stable and thereby curtail translation and decreaseproduction of the resultant protein. Likewise, AREs can be identifiedand removed or mutated to increase the intracellular stability and thusincrease translation and production of the resultant protein.

In one embodiment, the 3′ UTR of the viral genome ma.y include anoligo(dT) sequence for templated addition of a poly-A tail.

In one embodiment, the viral genome may include at least one miRNA seed,binding site or full sequence. micmRNAs (or miRNA or miR) are 19-25nucleotide noncoding RNAs that bind to the sites of nucleic acid targetsand down-regulate gene expression either by reducing nucleic acidmolecule stability or by inhibiting translation. A microRNA sequencecomprises a “seed” region, i.e.. a sequence in the region of positions2-8 of the mature microRNA, which sequence has perfect Watson-Crickcomplementarity to the miRNA target sequence of the nucleic acid.

In one embodiment, the viral genome may be engineered to include, alteror remove at least one .miRNA binding site, sequence or seed region.

Any UTR from any gene known in the art may be incorporated into theviral genome of the AAV particle. These UTRs, or portions thereof, maybe placed in the same orientation as in the gene from which they wereselected or they may be altered in orientation or location. In oneembodiment, the UTR used in the viral genome of the AAV particle may beinverted, shortened, lengthened, made with one or more other 5′ UTRs or3′ UTRs known in the art. As used herein, the term “altered” as itrelates to a UTR, means that the UTR has been changed in some way inrelation to a reference sequence. For example, a 3′ or 5′ UTR may bealtered relative to a wild type or native UTR by the change inorientation or location as taught above or may be altered by theinclusion of additional nucleotides, deletion of nucleotides, swappingor transposition of nucleotides.

In one embodiment, the viral genome of the AAV particle comprises atleast one artificial UTRs which is not a variant of a wild type UTR.

In one embodiment, the viral genome of the AAV particle comprises UTRswhich have been selected from a family of transcripts whose proteinsshare a common function, structure, feature or property.

Viral Genome Component: Polyadenylation Sequence

In one embodiment, the viral genome of the AAV particles of the presentdisclosure comprise at least one polyadenylation sequence. The viralgenome of the AAV particle may comprise a polyadenylation sequencebetween the 3′ end of the payload coding sequence and the 5′ end of the3′ITR.

In one embodiment, the polyadenylation sequence or “polyA sequence” mayrange from absent to about 500 nucleotides in length. Thepolyadenylation sequence may be, but is not limited to, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251,252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265,266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279,280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293,294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307,308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321,322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335,336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349,350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363,364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377,378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391,392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405,406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419,420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433,434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447,448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461,462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475,476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489,490, 491, 492, 493, 494, 495, 496, 497, 498, 499, and 500 nucleotides inlength.

In one embodiment, the polyadenylation sequence is 50-100 nucleotides inlength.

In one embodiment, the polyadenylation sequence is 50-150 nucleotides inlength.

In one embodiment, the polyadenylation sequence is 50-160 nucleotides inlength.

In one embodiment, the polyadenylation sequence is 50-200 nucleotides inlength.

In one embodiment, the polyadenylation sequence is 60-100 nucleotides inlength.

In one embodiment, the polyadenylation sequence is 60-150 nucleotides inlength.

In one embodiment, the polyadenylation sequence is 60-160 nucleotides inlength.

In one embodiment, the polyadenylation sequence is 60-200 nucleotides inlength.

In one embodiment, the polyadenylation sequence is 70-100 nucleotides inlength.

In one embodiment, the polyadenylation sequence is 70-150 nucleotides inlength.

In one embodiment, the polyadenylation sequence is 70-160 nucleotides inlength.

In one embodiment, the polyadenylation sequence is 70-200 nucleotides inlength.

In one embodiment, the polyadenylation sequence is 80-100 nucleotides inlength.

In one embodiment, the polyadenylation sequence is 80-150 nucleotides inlength.

In one embodiment, the polyadenylation sequence is 80-160 nucleotides inlength.

In one embodiment, the polyadenylation sequence is 80-200 nucleotides inlength.

In one embodiment, the polyadenylation sequence is 90-100 nucleotides inlength.

In one embodiment, the polyadenylation sequence is 90-150 nucleotides inlength.

In one embodiment, the polyadenylation sequence is 90-160 nucleotides inlength.

In one embodiment, the polyadenylation sequence is 90-200 nucleotides inlength.

In one embodiment, the AAV particle comprises a nucleic acid sequenceencoding an siRNA molecule may be located upstream of thepolyadenylation sequence in an expression vector, Further, the AAVparticle comprises a nucleic acid sequence encoding an siRNA moleculemay be located downstream of a promoter such as, but not limited to,CMV, U6, CAG, CBA or a CBA promoter with a SV40 intron or a human betaglobin intron in an expression vector. As a non-limiting example, theAAV particle comprises a nucleic acid sequence encoding an siRNAmolecule may be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30or more than 30 nucleotides downstream from the promoter and/or upstreamof the polyadenylation sequence in an expression vector. As anothernon-limiting example, the AAV particle comprises a nucleic acid sequenceencoding an siRNA molecule may be located within 1-5, 1-10, 1-15, 1-20,1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30,15-20, 15-25, 15-30, 20-25, 20-30 or 25-30 nucleotides downstream fromthe promoter and/or upstream of the polyadenylation sequence in anexpression vector. As a non-limiting example, the AAV particle comprisesa nucleic acid sequence encoding an siRNA molecule may be located withinthe first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or morethan 25% of the nucleotides downstream from the promoter and/or upstreamof the polyadenylation sequence in an expression vector. As anothernon-limiting example, the AAV particle comprises a nucleic acid sequenceencoding an siRNA molecule may be located with the first 1-5%, 1-10%,1-15%, 1-20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%, 10-15%, 10-20%, 10-25%,15-20%, 15-25%, or 20-25% downstream from the promoter and/or upstreamof the polyadenylation sequence in an expression vector.

In one embodiment, the AAV particle comprises a rabbit globinpolyadenylation (polyA) signal sequence (rBGpA),

In one embodiment, the AAV particle comprises a human growth hormonepolyadenylation (polyA) signal sequence.

Viral Genome Component: Introns

In one embodiment, the payload region comprises at least one element toenhance the expression such as one or more introns or portions thereof.Non-limiting examples of introns include, MVM (67-97 bps), F.IXtruncated intron 1 (300 bps), β-globin SD/immunoalobulin heavy chainsplice acceptor (250 bps), adenovirus splice donor/immunoglobin spliceacceptor (500 bps), SV40 late splice donor/splice acceptor (19S/16S)(180 bps) and hybrid adenovirus splice donor/IgG splice acceptor (230bps).

In one embodiment, the intron or intron portion may be 100-500nucleotides in length. The intron may have a length of 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440,450, 460, 470, 480, 490 or 500. The intron may have a length between80-100, 80-120, 80-140, 80-160, 80-180, 80-200, 80-250, 80-300, 80-350,80-400, 80-450, 80-500, 200-300, 200-400, 200-500, 300-400, 300-500, or400-500.

In one embodiment, the AAV viral genome may comprise a promoter such as,but not limited to, CMV or U6. As a non-limiting example, the promoterfor the AAV comprising the nucleic acid sequence for the siRNA moleculesof the present disclosure is a CMV promoter. As another non-limitingexample, the promoter for the AAV comprising the nucleic acid sequencefor the siRNA molecules of the present disclosure is a U6 promoter.

In one embodiment, the AAV viral genome may comprise a CMV promoter.

In one embodiment, the AAV viral genome may comprise a U6 promoter.

In one embodiment, the AAV viral genome may comprise a CMV and a U6promoter.

In one embodiment, the AAV viral genome may comprise a H1 promoter.

In one embodiment, the AAV viral genome may comprise a CBA promoter.

In one embodiment, the encoded siRNA molecule may be located downstreamof a promoter in an expression vector such as, but not limited to, CMV,U6, HI, CBA, CAG, or a CBA promoter with an intron such as SV40 orothers known in the art. Further, the encoded siRNA molecule may also belocated upstream of the polyadenylation sequence in an expressionvector. As a non-limiting example, the encoded siRNA. molecule may belocated within 1, 2, 3, 4, 5, 6, 7, S, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20.21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30nucleotides downstream from the promoter and/or upstream of thepolyadenylation sequence in an expression vector. As anothernon-limiting example, the encoded siRNA. molecule may be located within1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-.15,10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30 or 25-30nucleotides downstream from the promoter and/or upstream of thepolyadenylation sequence in an expression vector. As a non-limitingexample, the encoded siRNA molecule may be located within the first 1%,2%, 3%, 4% 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or more than 25% ofthe nucleotides downstream from the promoter and/or upstream of thepolyadenylation sequence in an expression vector. As anothernon-limiting example, the encoded siRNA. molecule may be located withthe first 1-5%, 1-10%, 1-15%, 1-20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%,10-15%, 10-20%, 10-25%, 15-20%, 15-25%, or 20-25% downstream from thepromoter and/or upstream of the polyadenylation sequence in anexpression vector.

Viral Genome Component: Filler Sequence

In one embodiment, the viral genome comprises one or more fillersequences.

In one embodiment, the viral genome comprises one or more fillersequences in order to have the length of the viral genome be the optimalsize for packaging. As a non-limiting example, the viral genomecomprises at least one filler sequence in order to have the length ofthe viral genome be about 2.3 kb. As a non-limiting example, the viralgenome comprises at least one filler sequence in order to have thelength of the viral genome be about 4.6 kb.

In one embodiment, the viral genome comprises one or more fillersequences in order to reduce the likelihood that a hairpin structure ofthe vector genome (e.g., a modulatory polynucleotide described herein)may be read as an inverted tenninal repeat (ITR) during expressionand/or packaging. As a non-limiting example, the viral genome comprisesat least one filler sequence in order to have the length of the viralgenome be about 2.3 kb. As a non-limiting example, the viral genomecomprises at least one filler sequence in order to have the length ofthe viral genome be about 4.6 kb.

In one embodiment, the viral genome is a single stranded (ss) viralgenome and comprises one or more filler sequences which have a lengthabout between 0.1 kb-3.8 kb, such as, but not limited to, 0.1 kb, 0.2kb, 0.3 kb, 0.4 kb, 0.5 kh, 0.6 kb, 0.7 kb, 0.8 kb, 0.9 kb, 1 kb, 1.1kb, 1.2 kb, 1,3 kb, 1.4 kb, 1.5 kb, 1.6 kb, 1.7 kb, 1.8 kb, 1,9 kb, 2kb, 2.1 kb, 2.2 kb, 2.3 kb, 2.4 kb, 2.5 kb, 2.6 kb, 2.7 kb, 2.8 kb, 2.9kb, 3 kb, 3.1 kb, 3.2 kb, 3.3 kb, 3.4 kb, 3.5 kb, 3.6 kb, 3.7 kb, or 3.8kb. As a non-limiting example, the total length filler sequence in thevector genome is 3.1 kb. As a non-limiting example, the total lengthfiller sequence in the vector genome is 2.7 kb. As a non-limitingexample, the total length filler sequence in the vector genome is 0.8kb. As a non-limiting example, the total length filler sequence in thevector genome is 0.4 kb. As a non-limiting example, the length of eachfiller sequence in the vector genome is 0.8 kb. As a non-limitingexample, the length of each filler sequence in the vector genome is 0.4kb.

In one embodiment, the viral genome is a self-complementary (sc) viralgenome, and comprises one or more filler sequences which have a lengthabout between 0.1 kb-1.5 kb, such as, but not limited to, 0.1 kb, 0.2kb, 0.3 kb, 0.4 kb, 0.5 kb, 0.6 kb, 0,7 kb, 0.8 kb, 0.9 kb, 1 kb, 1.1kb, 1.2 kb, 1.3 kb, 1.4 kb, or 1.5 kb. As a non-limiting example, thetotal length filler sequence in the vector genome is 0.8 kb. As anon-limiting example, the total length filler sequence in the vectorgenome is 0.4 kb. As a non-limiting example, the length of each fillersequence in the vector genome is 0.8 kb. As a non-limiting example, thelength of each filler sequence in the vector genome is 0.4 kb.

In one embodiment, the viral genome comprises any portion of a fillersequence. The viral genome may comprise 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 8c%, 90%, 95%, or 99% of a filler sequence.

In one embodiment, the viral genome is a single stranded (ss) viralgenome and comprises one or more filler sequences in order to have thelength of the viral genome be about 4.6 kb. As a non-limiting example,the viral genome comprises at least one filler sequence and the fillersequence is located 3′ to the. 5′ ITR sequence. As a non-limitingexample, the viral genome comprises at least one filler sequence and thefiller sequence is located 5′ to a promoter sequence. As a non-limitingexample, the viral genome comprises at least one filler sequence and thefiller sequence is located 3′ to the polyadenylation signal sequence. Asa non-limiting example, the viral genome comprises at least one fillersequence and the filler sequence is located 5′ to the 3′ ITR sequence.As a non-limiting example, the viral genome comprises at least onefiller sequence, and the filler sequence is located between two intronsequences. As a non-limiting example, the viral genome comprises atleast one filler sequence, and the filler sequence is located within anintron sequence. As a non-limiting example, the viral genome comprisestwo filler sequences, and the first filler sequence is located 3′ to the5′ ITR sequence and the second filler sequence is located 3′ to thepolyadenylation signal sequence. As a non-limiting example, the viralgenome comprises two filler sequences, and the first filler sequence islocated 5′ to a promoter sequence and the second filler sequence islocated 3′ to the polyadenylation signal sequence. As a non-limitingexample, the viral genome comprises two filler sequences, and the firstfiller sequence is located 3′ to the 5′ ITR sequence and the secondfiller sequence is located 5′ to the 5′ ITR sequence.

In one embodiment, the viral genome is a self-complementary (sc) viralgenome and comprises one or more filler sequences in order to have thelength of the viral genome be about 2.3 kb. As a non-limiting example,the viral genome comprises at least one filler sequence and the fillersequence is located 3′ to the 5′ ITR sequence. As a non-limitingexample, the viral genome comprises at least one filler sequence and thefiller sequence is located 5′ to a promoter sequence. As a non-limitingexample, the viral genome comprises at least one filler sequence and thefiller sequence is located 3′ to the polyadenylation signal sequence. Asa non-limiting example, the viral genome comprises at least one fillersequence and the filler sequence is located 5′ to the 3′ ITR. sequence.As a non-limiting example, the viral genome comprises at least onefiller sequence, and the filler sequence is located between two intronsequences. As a non-limiting example, the viral genome comprises atleast one filler sequence, and the filler sequence is located within anintron sequence. As a non-limiting example, the viral genome comprisestwo filler sequences, and the first filler sequence is located 3′ to the5′ ITR sequence and the second filler sequence is located 3′ to thepolyadenylation signal sequence. As a non-limiting example, the viralgenome comprises two filler sequences, and the first filler sequence islocated 5′ to a promoter sequence and the second filler sequence islocated 3′ to the polyadenylation signal sequence. As a non-limitingexample, the viral genome comprises two filler sequences, and the firstfiller sequence is located 3′ to the 5′ ITR sequence and the secondfiller sequence is located 5′ to the 5′ ITR sequence.

In one embodiment, the viral genome may comprise one or more fillersequences between one of more regions of the viral genome. In oneembodiment, the filler region may be located before a region such as,but not limited to, a payload region, an inverted terminal repeat (ITR),a promoter region, an intron region, an enhancer region, apolyadenylation signal sequence region, and/or an exon region. In oneembodiment, the filler region may be located after a region such as, butnot limited to, a payload region, an inverted terminal repeat (ITR), apromoter region, an intron region, an enhancer region, a polyadenylationsignal sequence region, and/or an exon region. In one embodiment, thefiller region may be located befbre and after a region such as, but notlimited to, a payload region, an inverted terminal repeat (ITR), apromoter region, an intron region, an enhancer region, a polyadenylationsignal sequence region, and/or an exon region.

In one embodiment, the viral genome may comprise one or more fillersequences which bifurcates at least one region of the viral genome. Thebifurcated region of the viral genome may comprise 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the of the region to the 5′of the filler sequence region. As a non-limiting example, the fillersequence may bifurcate at least one region so that 10% of the region islocated 5′ to the filler sequence and 90% of the region is located 3′ tothe filler sequence. As a non-limiting example, the filler sequence maybifurcate at least one region so that 20% of the region is located 5′ tothe filler sequence and 80% of the region is located 3′ to the fillersequence. As a non-limiting example, the filler sequence may bifurcateat least one region so that 30% of the region is located 5′ to thefiller sequence and 70% of the region is located 3′ to the fillersequence. As a non-limiting example, the filler sequence may bifurcateat least one region so that 40% of the region is located 5′ to thefiller sequence and 60% of the region is located 3′ to the fillersequence. As a non-limiting example, the filler sequence may bifurcateat least one region so that 50% of the region is located 5′ to thefiller sequence and 50% of the region is located 3′ to the fillersequence. As a non-limiting example, the filler sequence may bifurcateat least one region so that 60% of the region is located 5′ to thefiller sequence and 40% of the region is located 3′ to the fillersequence. As a non-limiting example, the filler sequence may bifurcateat least one region so that 70% of the region is located 5′ to thefiller sequence and 30% of the region is located 3′ to the fillersequence. As a non-limiting example, the filler sequence may bifurcateat least one region so that 80% of the region is located 5′ to thefiller sequence and 20% of the region is located 3′ to the fillersequence. As a non-limiting example, the filler sequence may bifurcateat least one region so that 90% of the region is located 5′ to thefiller sequence and 10% of the region is located 3′ to the fillersequence.

In one embodiment, the viral genome comprises a filler sequence afterthe 5′ ITR.

In one embodiment, the viral genome comprises a filler sequence afterthe promoter region. In one embodiment, the viral genome comprises afiller sequence after the payload region. In one embodiment, the viralgenome comprises a filler sequence after the intron region. In oneembodiment, the viral genome comprises a filler sequence after theenhancer region. In one embodiment, the viral genome comprises a fillersequence after the polyadenylation signal sequence region. In oneembodiment, the viral genome comprises a filler sequence after the exonregion.

In one embodiment, the viral genome comprises a filler sequence beforethe promoter region. In one embodiment, the viral genome comprises afiller sequence before the payload region. In one embodiment, the viralgenome comprises a filler sequence before the intron region. In oneembodiment, the viral genome comprises a filler sequence before theenhancer region. In one embodiment, the viral genome comprises a fillersequence before the polyadenylation signal sequence region. In oneembodiment, the viral genome comprises a filler sequence before the exonregion.

In one embodiment, the viral genome comprises a filler sequence beforethe 3′ ITR.

In one embodiment, a filler sequence may be located between two regions,such as, but not limited to, the 5′ ITR and the promoter region. In oneembodiment, a filler sequence may be located between two regions, suchas, but not limited to, the 5′ ITR and the payload region. In oneembodiment, a filler sequence may be located between two regions, suchas, but not limited to, the 5′ ITR and the intron region. In oneembodiment, a filler sequence may be located between two regions, suchas, but not limited to, the 5′ FUR and the enhancer region. in oneembodiment, a filler sequence may be located between two regions, suchas, but not limited to, the 5′ ITR and the polyadenylation signalsequence region,

In one embodiment, a filler sequence may be located between two regions,such as, but not limited to, the 5′ ITR and the exon region.

In one embodiment, a filler sequence may be located between two regions,such as, but not limited to, the promoter region and the payload region.In one embodiment, a filler sequence may be located between two regions,such as, but not limited to, the promoter region and the intron region.In one embodiment, a filler sequence may be located between two regions,such as, but not limited to, the promoter region and the enhancerregion. In one embodiment, a filler sequence may be located between tworegions, such as, but not limited to, the promoter region and thepolyadenylation signal sequence region. In one embodiment, a fillersequence may be located between two regions, such as, but not limitedto, the promoter region and the exon region. In one embodiment, a fillersequence may be located between two regions, such as, but not limitedto, the promoter region and the 3′ ITR.

In one embodiment, a filler sequence may be located between two regions,such as, but not limited to, the payload region and the intron region.In one embodiment, a filler sequence may be located between two regions,such as, but not limited to, the payload region and the enhancer region.In one embodiment, a filler sequence may be located between two regions,such as, but not limited to, the payload region and the polyadenylationsignal sequence region. In one embodiment, a filler sequence may belocated between two regions, such as, but not limited to, the payloadregion and the exon region.

In one embodiment, a filler sequence may be located between two regions,such as, but not limited to, the payload region and the 3′ ITR.

In one embodiment, a filler sequence may be located between two regions,such as, but not limited to, the intron region and the enhancer region.In one embodiment, a filler sequence may be located between two regions,such as, but not limited to, the intron region and the polyadenylationsignal sequence region. In one embodiment, a filler sequence may belocated between two regions, such as, but not limited to, the intronregion and the exon region. In one embodiment, a filler sequence may belocated between two regions, such as, but not limited to, the intronregion and the 3′ ITR. In one embodiment, a filler sequence may belocated between two regions, such as, but not limited to, the enhancerregion and the polyadenylation signal sequence region. In oneembodiment, a filler sequence may be located between two regions, suchas, but not limited to, the enhancer region and the exon region. In oneembodiment, a filler sequence may be located between two regions, suchas, but not limited to, the enhancer region and the 3′ ITR.

In one embodiment, a filler sequence may be located between two regions,such as, but not limited to, the polyadenylation signal sequence regionand the exon region. In one embodiment, a filler sequence may be locatedbetween two regions, such as, but not limited to, the polyadenylationsignal sequence region and the 3′ ITR.

In one embodiment, a filler sequence may be located between two regions,such as, but not limited to, the exon region and the 3′ ITR.

Payloads

The AAV particles of the present disclosure comprise at least onepayload region. As used herein, “payload” or “payload region” refers toone or more polynucleotides or polynucleotide regions encoded by orwithin a viral genome or an expression product of such polynucleotide orpolynucleotide region, a transgene, a polynucleotide encoding apolypeptide or multi-polypeptide or a modulatory nucleic acid orregulatory nucleic acid. Payloads of the present disclosure typicallyencode modulatory polynucleotides or fragments or variants thereof.

The payload region may be constructed in such a way as to reflect aregion similar to or mirroring the natural organization of an mRNA.

The payload region may comprise a combination of coding and non-codingnucleic acid sequences.

In some embodiments, the AAV payload region may encode a coding ornon-coding RNA.

In one embodiment, the AAV particle comprises a viral genome with apayload region comprising nucleic acid sequences encoding a siRNA, miRNAor other RNAi agent. In such an embodiment, a viral genome encoding morethan one polypeptide may be replicated and packaged into a viralparticle. A target cell transduced with a viral particle may express theencoded siRNA, miRNA or other RNAi agent inside a single cell.

Modulatory Polynucleotides

In one embodiment, modulatory polynucleotides, e.g., RNA or DNAmolecules, may be used to treat neurodegenerative disease, inparticular, amyotrophic lateral sclerosis (ALS). As used herein, a“modulatory polynucleotide” is any nucleic acid sequence(s) whichfunctions to modulate (either increase or decrease) the level or amountof a target gene, e.g., mRNA or protein levels.

In one embodiment, the modulatory polynucleotides may comprise at leastone nucleic acid sequence encoding at least one siRNA molecule. Thenucleic acids may, independently if there is more than one, encode 1, 2,3, 4, 5, 6, 7, 8, 9, or more than 9 siRNA molecules.

In one embodiment, the molecular scaffold may be located downstream of aCMV promoter, fragment or variant thereof.

In one embodiment, the molecular scaffold may be located downstream of aCBA promoter, fragment or variant thereof.

In one embodiment, the molecular scaffold may be a natural pri-miRNAscaffold located downstream of a CMV promoter. As a non-limitingexample, the natural pri-miRNA scaffold is derived from the human miR155scaffold.

In one embodiment, the molecular scaffold may be a natural pri-miRNAscaffold located downstream of a CBA promoter.

In one embodiment, the selection of a molecular scaffold and modulatorypolynucleotide is determined by a method of comparing modulatorypolynucleotides in pri-miRNA (see e.g., the method described byMiniarikova et al. Design, Characterization, and Lead Selection ofTherapeutic miRNAs Targeting Huntingtin ibr Development of Gene Therapyfor Huntington's Disease. Molecular Therapy-Nucleic Acids (2016) 5, e297and International Publication No. WO2016102664; the contents of each ofwhich are herein incorporated by reference in their entireties). Toevaluate the activities of the modulatory polynucleotides, the molecularscaffold used which may be used is a human pri-miRNA scaffold (e.g.,miR155 scaffold) and the promoter may be CMV. The activity may bedetermined in vitro using HEK293T cells and a reporter (e.g.,Luciferase).

In order to evaluate the optimal molecular scaffold for the modulatorypolynucleotide, the modulatory polynucleotide is used in pri-miRNA.scaffolds with a CAG promoter. The constructs are co-transfected with areporter (e.g., luciferase reporter) at 50 ng. Constructs with greaterthan 80% knockdown at 50 ng co-transfection are considered efficient. Inone aspect, the constructs with strong guide-strand activity arepreferred. The molecular scaffolds can be processed in HEK293T cells byNGS to determine guide-passenger ratios, and processing variability.

To evaluate the molecular scaffolds and modulatory polynucleotides invivo the molecular scaffolds comprising the modulatory polynucleotidesare packaged in AAV (e.g., the serotype may be AAV5 (see e.g., themethod and constructs described in WO2015060722, the contents of whichare herein incorporated by reference in their entirety)) andadministered to an in vivo model and the guide-passenger ratios, 5′ and3′ end processing, ratio of guide to passenger strands, and knockdowncan be determined in different areas of the model (e.g., tissueregions).

In one embodiment, the selection of a molecular scaffold and modulatorypolynucleotide is determined by a method of comparing modulatorypolynucleotides in natural pri-miRNA and synthetic pri-miRNA. Themodulatory polynucleotide may, but it not limited to, targeting an exonother than exon 1. To evaluate the activities of the modulatorypolynucleotides, the molecular scaffold is used with a CBA promoter. Inone aspect, the activity may be determined in vitro using HEK293T cells,HeLa cell and a reporter (e,g., Luciferase) and knockdown efficientmodulatory polynucleotides showed SOD1 knockdown of at least 80% in thecell tested. Additionally, the modulatory polynucleotides which areconsidered most efficient showed low to no significant passenger strand(p-strand) activity. In another aspect, the endogenous SOD1 knockdownefficacy is evaluated by transfection in vitro using HEK293T cells, HeLacell and a reporter. Efficient modulatory polynucleotides show greaterthan 50% endogenous SOD1 knockdown. In yet another aspect, theendogenous SOD1 knockdown efficacy is evaluated in different cell types(e.g., HEK293, HeLa, primary astrocytes, U251 astrocytes, SH-SYSY neuroncells and fibroblasts from ALS patients) by infection (e.g., AAV2).Efficient modulatory polynucleotides show greater than 60% endogenousSOD1 knockdown.

To evaluate the molecular scaffolds and modulatory polynucleotides invivo the molecular scaffolds comprising the modulatory polynucleotidesare packaged in AAV and administered to an in vivo model and theguide-passenger ratios, 5′ and 3′ end processing, ratio of guide topassenger strands, and knockdown can be determined in different areas ofthe model (e.g., tissue regions). The molecular scaffolds can beprocessed from in vivo samples by NGS to determine guide-passengerratios, and processing variability.

In one embodiment, the modulatory polynucleotide is designed using atleast one of the following properties: loop variant, seedmismatch/bulge/wobble variant, stem mismatch, loop variant and vassalstem mismatch variant, seed mismatch and basal stem mismatch variant,stem mismatch and basal stem mismatch variant, seed wobble and basalstem wobble variant, or a stem sequence variant.

The present disclosure relates, in part, to RNA interfering (RNAi)induced inhibition of gene expression for treating neurodegenerativedisorders. Provided are siRNA duplexes or dsRNA that target SOD1 gene.Such siRNA duplexes or dsRNA can silence SOD1 gene expression in cells,for example, motor neurons, therefore, ameliorating symptoms of ALS suchas motor death and muscle atrophy. The SOD1 siRNA may be encoded inpolynucleotides of a recombinant AAV vector.

siRNA duplexes or dsRNA targeting a specific mRNA may be designed andsynthesized as part of a target SOD1 targeting polynucleotide in vitroand introduced into cells for activating RNAi process.

siRNA Molecules

The present disclosure relates to RNA interference (RNAi) inducedinhibition of gene expression for treating neurodegenerative disorders.Provided herein are siRNA duplexes or encoded dsRNA that target the geneof interest (referred to herein collectively as “siRNA molecules”). SuchsiRNA duplexes or encoded dsRNA can reduce or silence gene expression incells, such as but not limited to, medium spiny neurons, corticalneurons and/or astrocytes.

RNAi (also known as post-transcriptional gene silencing (PTGS),quelling, or co-suppression) is a post-transcriptional gene silencingprocess in which RNA molecules, in a. sequence specific manner, inhibitgene expression, typically by causing the destruction of specific mRNAmolecules. The active components of RNAi are short/small double strandedRNAs (dsRNAs), called small interfering RNAs (siRNAs), that typicallycontain 15-30 nucleotides (e.g., 19 to 25, 19 to 24 or 19-21nucleotides) and 2 nucleotide 3′ overhangs and that match the nucleicacid sequence of the target gene. These short RNA species may benaturally produced in vivo by Dicer-mediated cleavage of larger dsRNAsand they are functional in mammalian cells.

Naturally expressed small RNA molecules, named microRNAs (miRNAs),elicit gene silencing by regulating the expression of mRNAs. The miRNAscontaining RNA Induced Silencing Complex (RISC) targets mRNAs presentinga perfect sequence complementarity with nucleotides 2-7 in the 5′regionof the miRNA which is called the seed region, and other base pairs withits 3′region. miRNA mediated down regulation of gene expression may becaused by cleavage of the target mRNAs, translational inhibition of thetarget mRNAs, or mRNA decay. miRNA targeting sequences are usuallylocated in the 3′-UTR of the target mRNAs. A single miRNA may targetmore than 100 transcripts from various genes, and one mRNA may betargeted by different miRNAs.

siRNA duplexes or dsRNA targeting a specific mRNA may be designed and.synthesized in vitro and introduced into cells for activating RNAiprocesses. Elbashir et al. demonstrated that 21-nucleotide siRNAduplexes (termed small interfering RNAs) were capable of effectingpotent and specific gene knockdown without inducing immune response inmammalian cells (Elbashir S M et al., Nature, 2001, 411, 494-498). Sincethis initial report, post-transcriptional gene silencing by siRNAsquickly emerged as a powerful tool for genetic analysis in mammaliancells and has the potential to produce novel therapeutics.

RNAi molecules which were designed to target against a nucleic acidsequence that encodes poly-glutamine repeat proteins which causepoly-glutamine expansion diseases such as Huntington's Disease, aredescribed in U.S. Pat. No. 9,169,483 and 9,181,544 and InternationalPatent Publication No. WO2015179525, the content of each of which isherein incorporated by reference in their entirety. U.S. Pat. Nos.9,169,483 and 9,181,544 and International Patent Publication No.WO2015179525 each provide isolated RNA duplexes comprising a firststrand of RNA (e.g., 15 contiguous nucleotides) and second strand of RNA(e.g., complementary to at least 12 contiguous nucleotides of the firststrand) where the RNA duplex is about 15 to 30 base pairs in length. Thefirst strand of RNA and second strand of RNA may be operably linked byan RNA loop (˜4 to 50 nucleotides) to form a hairpin structure which maybe inserted into an expression cassette. Non-limiting examples of loopportions include SEQ ID NO: 9-14 of U.S. Pat. No. 9,169,483, the contentof which is herein incorporated by reference in its entirety.Non-limiting examples of strands of RNA which may be used, either fullsequence or part of the sequence, to form RNA duplexes include SEQ IDNO: 1-8 of U.S. Pat. No. 9,169,483 and SEQ ID NO: 1-11, 33-59, 208-210,213-215 and 218-221 of U.S. Pat. No. 9,181,544, the contents of each ofwhich is herein incorporated by reference in its entirety. Non-limitingexamples of RNAi molecules include SEQ ID NOs: 1-8 of U.S. Pat. No.9,169,483, SEQ ID NOs: 1-11, 33-59, 208-210, 213-215 and 218-221 of U.S.Pat. No. 9,181,544 and. SEQ NOs: 1, 6, 7, and 35-38 of InternationalPatent Publication No. WO2015179525, the contents of each of which isherein incorporated by reference in their entirety.

In vitro synthesized siRNA molecules may be introduced into cells inorder to activate RNAi. An exogenous siRNA duplex, when it is introducedinto cells, similar to the endogenous dsRNAs, can be assembled to formthe RNA Induced Silencing Complex (RISC), a multiunit complex thatinteracts with RNA sequences that are complementary to one of the twostrands of the siRNA duplex (i.e., the antisense strand). During theprocess, the sense strand (or passenger strand) of the siRNA is lostfrom the complex, while the antisense strand (or guide strand) of thesiRNA is matched with its complementary RNA. In particular, the targetsof siRiNA containing RISC complexes are mRNAs presenting a perfectsequence complementarity. Then, siRNA mediated gene silencing occurs bycleaving, releasing and degrading the target.

The siRNA duplex comprised of a sense strand homologous to the targetmRNA and an antisense strand that is complementary to the target mRNAoffers much more advantage in terms of efficiency for target RNAdestruction compared to the use of the single strand (ss)-siRNAs (e.g.antisense strand RNA or antisense oligonucleotides). In many cases, itrequires higher concentration of the ss-siRiNA to achieve the effectivegene silencing potency of the corresponding duplex.

Any of the foregoing molecules may be encoded by a viral genome.

Design and Sequences of siRNA Duplexes Targeting Gene of Interest

The present disclosure provides small interfering RNA (siRNA) duplexes(and modulatory polynucleotides encoding them) that target mRNA tointerfere with gene expression and/or protein production.

The encoded siRNA duplex of the present disclosure contains an antisensestrand and a sense strand hybridized together forming a duplexstructure, wherein the antisense strand is complementary to the nucleicacid sequence of the targeted gene, and wherein the sense strand ishomologous to the nucleic acid sequence of the targeted gene. In someaspects, the 5′end of the antisense strand has a 5′ phosphate group andthe 3′end of the sense strand contains a 3′hydroxyl group. In otheraspects, there are none, one or 2 nucleotide overhangs at the 3′end ofeach strand.

Some guidelines for designing siRNAs have been proposed in the art.These guidelines generally recommend generating a 19-nucleotide duplexedregion, symmetric 2-3 nucleotide 3′overhangs, 5′-phosphate and3′-hydroxyl groups targeting a region in the gene to be silenced. Otherrules that may govern siRNA sequence preference include, but are notlimited to, (i) A/U at the 5′ end of the antisense strand; (ii) G/C atthe 5′ end of the sense strand; (iii) at least five A/U residues in the5′ terminal one-third of the antisense strand; and (iv) the absence ofany GC stretch of more than 9 nucleotides in length. In accordance withsuch consideration, together with the specific sequence of a. targetgene, highly effective siRNA molecules essential for suppressingmammalian target gene expression may be readily designed.

According to the present disclosure, siRNA molecules (e.g., siRNAduplexes or encoded dsRNA) that target the gene of interest aredesigned. Such siRNA molecules can specifically, suppress geneexpression and protein production. In some aspects, the siRNA moleculesare designed and used to selectively “knock out” gene variants in cells,i.e., mutated transcripts. In some aspects, the siRNA molecules aredesigned and used to selectively “knock down” gene variants in cells. Inother aspects, the siRNA molecules are able to inhibit or suppress boththe wild type and mutated version of the gene of interest.

In one embodiment, an siRNA molecule of the present disclosure comprisesa sense strand and a complementary antisense strand in which bothstrands are hybridized together to form a duplex structure. Theantisense strand has sufficient complementarity to the target mRNAsequence to direct target-specific RNAi, i.e., the siRNA molecule has asequence sufficient to trigger the destruction of the target mRNA by theRNAi machinery or process.

In one embodiment, an siRNA molecule of the present disclosure comprisesa sense strand and a complementary antisense strand in which bothstrands are hybridized together to form a duplex structure and where thestart site of the hybridization to the mRNA is between nucleotide 10 and1000 on the target mRNA sequence. As a non-limiting example, the startsite may be between nucleotide 10-20, 20-30, 30-40, 40-50, 60-70, 70-80,80-90, 90-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400,400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-70, 750-800,800-850, 850-900, 900-950, 950-1000, on the target mRNA sequence. As yetanother non-limiting example, the start site may be nucleotide 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26. 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380,381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394,395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408,409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422,423, 424, 425, 426, 427, 428, 429, 430, 431 432, 433, 434, 435, 436,437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450,451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464,465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478,479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492,493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506,507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520,521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534,535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548,549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562,563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576,577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590,591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604,605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618,619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632,633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646,647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660,661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674,675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688,689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702,703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716,717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730,731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744,745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758,759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771 772,773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786,787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800,801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814,815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828,829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842,843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856,857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870,871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884,885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898,899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912,913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926,927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940,941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954,955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968,969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982,983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996,997, 998, 999, and 1000 on the target mRNA sequence.

In some embodiments, the antisense strand and target mRNA sequences have100% complementarity. The antisense strand may be complementary to anypart of the target mRNA sequence.

In other emboditrtents, the antisense strand and target snRNA sequencescomprise at least one mismatch. As a non-limiting example, the antisensestrand and the target mRNA sequence have at least 30%, 40%, 50%, 60%,70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% or at least 20-30%, 20-40%, 20-50%,20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-99%, 30-40%, 30-50%, 30-60%,30-70%, 30-80%, 30-90%, 30-95%, 30-99%, 40-50%, 40-60%, 40-70%, 40-80%40-90% 40-95% 40-99%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%,60-70%, 60-80%, 60-90%, 60-95%, 60-99%, -80%, 70-90%, 70-95%, 70-99%,80-90%, 80-95%, 80-99%, 90-95%, 90-99% or 95-99% complementarity.

In one embodiment. an siRNA or dsRNA includes at least two sequencesthat are complementary to each other.

According to the present disclosure, the siRNA molecule has a lengthfrom about 10-50 or more nucleotides, i.e., each strand comprising 10-50nucleotides nucleotide analogs). Preferably, the siRNA molecule has alength from about 15-30, 15, 16. 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27. 28, 29, or 30 nucleotides in each strand, wherein one of the strandsis sufficiently complementarity to a target region. In one embodiment,each strand of the siRNA molecule has a length from about 19 to 25, 19to 24 or 19 to 21 nucleotides. In one embodiment, at least one strand ofthe siRNA molecule is 19 nucleotides in length. In one embodiment, atleast one strand of the siRNA molecule is 20 nucleotides in length. Inone embodiment, at least one strand of the siRNA molecule is 21nucleotides in length. In one embodiment, at least one strand of thesiRNA molecule is 22 nucleotides in length. In one embodiment, at leastone strand of the siRNA molecule is 23 nucleotides in length. In oneembodiment, at least one strand of the siRNA molecule is 24 nucleotidesin length. In one embodiment, at least one strand of the siRNA moleculeis 25 nucleotides in length.

In some embodiments, the siRNA molecules of the present disclosure canbe synthetic RNA duplexes comprising about 19 nucleotides to about 25nucleotides, and two overhanging nucleotides at the 3′-end. In someaspects, the siRNA molecules may be unmodified RNA molecules. In otheraspects, the siRNA molecules may contain at least one modifiednucleotide, such as base, sugar or backbone modifications.

In one embodiment, the siRNA molecules of the present disclosure maycomprise an antisense sequence and a sense sequence, or a fraament orvariant thereof. As a non-limiting example, the antisense sequence andthe sense sequence have at least 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%,20-80%, 20-90%, 20-95%, 20-99%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%,30-90%, 30-95%, 30-99%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%,40-99%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%, 60-70%, 60-80%,60-90%, 60-95%, 60-99%, 70-80%, 70-90%, 70-95%, 70-99%, 80-90%, 80-95%,80-99%, 90-95%, 90-99% or 95-99% complementarity.

In other embodiments, the siRNA molecules of the present disclosure canbe encoded in plasmid vectors, AAV particles, viral genome or othernucleic acid expression vectors for delivery to a cell.

DNA expression plasm ids can be used to stably express the siRNAduplexes or dsRNA of the present disclosure in cells and achievelong-term inhibition of the target gene expression. In one aspect, thesense and antisense strands of a siRNA duplex are typically linked by ashort spacer sequence leading to the expression of a stem-loop structuretermed short hairpin RNA (shRNA). The hairpin is recognized and cleavedby Dicer, thus generating mature siRNA molecules.

According to the present disclosure, AAV particles comprising thenucleic acids encoding the siRNA molecules targeting the mRNA areproduced, the AAV serotypes may be any of the serotypes listed herein.Non-limiting examples of the AAV serotypes include, AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11,AAV12, AAVrh8, AAVrh10, AAV-DJ8, AAV-DJ, AAV-PHP.A, AAV-PIP.B,AAVPHP.B2, AAVPHP.B3, AAVPHP.N/PHP.B-DGT, AAVPH.P.B-EST, AAVPHP.B-GGT,AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T,AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP,AAVPHR.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHR.B-SGN, AAVPHP.B-EGT,AAVPHP.B-DST, AAVPHR.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP,AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHR.B-TTP, AAVPHP.S/G2A12, AAVG2A15/G2A3,AAVG2B4, AAVG2B5, and variants thereof.

In some embodiments, the siRNA duplexes or encoded dsRNA of the presentdisclosure suppress (or degrade) the target mRNA. Accordingly, the siRNAduplexes or encoded dsRNA can be used to substantially inhibit the geneexpression in a cell, for example a neuron. In some aspects, theinhibition of the gene expression refers to an inhibition by at leastabout 20%, preferably by at least about 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84/%, 85%, 90%, 95%, 99% and 100%, or atleast 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%,20-100%, 30-40%, 30-45%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%,30-100%, 35-45%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%,40-100%, 45-50%, 45-55%, 50-60%, 50-70%, 50-75%, 50-80%, 50-90%, 50-95%,50-100%, 55-65%, 57-68%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%,70-80%, 70-85%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%,85-99%, 90-95%, 90-100% or 9.5-100%, Accordingly, the protein product ofthe targeted gene may be inhibited by at least about 20%, preferably byat least about 30%, 31%, 32%, 33%, 34%. 35%, 36%, 37%. 38%, 39%, 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58% 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 90%, 95%, 99% and 100%, or at least 20-30%, 20-40%,20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-45%,30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 35-45%, 40-50%,40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 45-50%, 45-55%, 50-60%,50-70%, 50-75%, 50-80%, 50-90%, 50-95%, 50-100%, 55-65%, 57-68%, 60-70%,60-80%, 60-90%. 60-95%. 60-100%, 70-80%, 70-85%, 70-90%, 70-95%,70-100%, 80-90%, 80-95%, 80-100%, 85-99%, 90-95%, 90-100% or 95-100%. Asa non-limiting example, the inhibition may be 30-40%. As a non-limitingexample, the inhibition may be 30-45%. As a non-limiting example, theinhibition may be 35-45%. As a non-limiting example, the inhibition maybe greater than 50%. As a non-limiting example, the inhibition may be50-60%. As a non-limiting example, the inhibition may be greater than60%. As a non-limiting example, the inhibition may be 50-75%. As anon-limiting example, the inhibition may be 55-65%. As a non-limitingexample, the inhibition may be 57-68%. As a non-limiting example, theinhibition may be 70-80%. As a non-limiting example, the inhibition maybe 70-85%. As a non-limiting example, the inhibition may be 85-99%. As anon-limiting example, the inhibition may be 35%. As a non-limitingexample, the inhibition may be 36%. As a non-limiting example, theinhibition may be 40%. As a non-limiting example, the inhibition may be41%. As a non-limiting example, the inhibition may be 43%. As anon-limiting example, the inhibition may be 45%. As a non-limitingexample, the inhibition may be 49%. As a non-limiting example, theinhibition may be 62%. As a non-limiting example, the inhibition may be64%. As a non-limiting example, the inhibition may be 74%. As anon-limiting example, the inhibition may be 77%. As a non-limitingexample, the inhibition may be 84%. As a non-limiting example, theinhibition may be 87%. As a non-limiting example, the inhibition may be95%. As a non-limiting example, the inhibition may be 99%. As anon-limiting example, the inhibition may be 100%.

In one embodiment, the siRNA duplexes or encoded dsRNA of the presentdisclosure suppress (or degrade) the target mRNA in spinal cord motorneurons. In some aspects, the inhibition of the gene expression refersto suppression of at least about 20%, preferably by at least about 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42% 43%, 44%,45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% 70%, 71, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 95%,99% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%,20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-45%, 30-50%, 30-60%, 30-70%,30-80%, 30-90%, 30-95%, 30-100%, 35-45%, 40-50%, 40-60%, 40-70%, 40-80%,40-90%, 40-95%, 40-100%, 45-50%, 45-55%, 50-60%, 50-70%, 50-75%, 50-80%,50-90%, 50-95%, 50-100%, 55-65%, 57-68%, 60-70% 60-80%, 60-90%, 60-95%,60-100%, 70-80%, 70-85%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%,80-100%, 85-99%, 90-95%, 90-100% or 95-100%. Accordingly, the proteinproduct of the targeted gene may be inhibited by at least about 20%,preferably by at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45% 46%, 47%, 48% 49%, 50%, 51% 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 90%, 95%, 99% and 100%, or at least 20-30%,20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%,30-45%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%,35-459%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%,45-50%, 45-55%, 50-60%, 50-70%, 50-75%, 50-80%, 50-90%, 50-95%, 50-100%,55-65%, 57-68%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%,70-8590, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 85-99%,90-95%, 90-100% or 95-100%. As a non-limiting example, the suppressionmay be 30-45%. As a non-limiting example, the suppression may be 35-45%.As a non-limiting example, the suppression may be greater than 50%. As anon-limiting example, the suppression may be greater than 60%. As anon-limiting example, the suppression may be 50-60%. As a non-limitingexample, the suppression may be 55-65%. As a non-limiting example, thesuppression may be 50-75%. As a non-limiting example, the suppressionmay be 57-68%. As a non-limiting example, the suppression may be 70-80%.As a non-limiting example, the suppression may be 70-85%. As anon-limiting example, the suppression may be 85-99%. As a non-limitingexample, the suppression may be 35%. As a non-limiting example, thesuppression may be 36%. As a non-limiting example, the suppression maybe 10%. As a non-limiting example, the suppression may be 41%, As anon-limiting example, the suppression may be 43%. As a non-limitingexample, the suppression may be 45%. As a non-limiting example, thesuppression may be 49%. As a non-limiting example, the suppression maybe 62%. As a non-limiting example, the suppression may be 64%. As anon-limiting example, the suppression may be 74%. As a non-limitingexample, the suppression may be 77%. As a non-limiting example, thesuppression may be 84%. As a non-limiting example, the suppression maybe 87%. As a non-limiting example, the suppression may be 95%. As anon-limiting example, the suppression may be 99%. As a non-limitingexample, the suppression may be 100%.

In one embodiment, the siRNA duplexes or encoded dsRNA of the presentdisclosure suppress (or degrade) the target mRNA in spinal cord motorneurons by 78%.

In one embodiment, the siRNA duplexes or encoded dsRNA of the presentdisclosure suppress (or degrade) the target mRNA in spinal cord motorneurons by 45-55%.

In one embodiment, the siRNA duplexes or encoded dsRNA of the presentdisclosure suppress (or degrade) the target mRNA in vg+ cells of motorneuron morphology. In some aspects, the inhibition of the geneexpression refers to an inhibition by at least about 20%, preferably byat least about 30%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 90%,95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 70-70%,20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%,30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%,40-100%, 45-50%, 45-55%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%,50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%,70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100%.Accordingly, the protein product of the targeted gene may be inhibitedby at least about 20%, preferably by at least about 30%, 40%, 41%, 42%,43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 90%, 95% and 100%, or at least 20-30%,20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90©′0 20-95%, 20-100%,30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%,40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 45-50%, 45-55%, 50-60%,50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%,60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%,90-95%, 90-100% or 95-100%.

In one embodiment, the siRNA duplexes or encoded dsRNA of the presentdisclosure suppress (or degrade) the target mRNA in vg+ cells of motorneuron morphology by 53%.

In one embodiment, the siRNA molecules comprise a miRNA seed match forthe target located in the guide strand. In another embodiment, the siRNAmolecules comprise a miRNA seed match for the target located in thepassenger strand, In yet another embodiment, the siRNA duplexes orencoded dsRNA targeting the gene of interest do not comprise a seedmatch for the target located in the guide or passenger strand.

In one embodiment, the siRNA duplexes or encoded dsRNA targeting thegene of interest may have almost no significant full-length off targeteffects for the guide strand. In another embodiment, the siRNA duplexesor encoded dsRNA targeting the gene of interest may have almost nosignificant full-length off target effects for the passenger strand. ThesiRNA duplexes or encoded dsRNA targeting the gene of interest may haveless than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 1-5%, 2-6%, 3-7%, 4-8%, 5-9%,5-10%, 6-10%, 5-15%, 5-20%, 5-25% 5-30%, 10-20%, 10-30%, 10-40%, 10-50%,15-30%, 15-40%, 15-45%, 20-40%, 20-50%, 25-50%, 30-40%, 30-50%, 35-50%,40-50%, 45-50% full-length off target effects for the passenger strand.In yet another embodiment, the siRNA duplexes or encoded dsRNA targetingthe gene of interest may have almost no significant full-length offtarget effects for the guide strand or the passenger strand. The siRNAduplexes or encoded dsRNA targeting the gene of interest may have lessthan 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 1-5%, 2-6%, 3-7%, 4-8%, 5-9%, 5-10%,6-10%, 5-15%, 5-20%, 5-25% 5-30%, 10-20%, 10-30%, 10-40%, 10-50%,15-30%, 15-40%, 15-45%, 20-40%, 20-50%, 25-50%, 30-40%, 30-50%, 35-50%,40-50%, 45-50% full-length off target effects for the guide or passengerstrand.

In one embodiment, the siRNA duplexes or encoded dsRNA targeting thegene of interest may have high activity in vitro. In another embodiment,the siRNA molecules may have low activity in vitro. In yet anotherembodiment, the siRNA duplexes or dsRNA. targeting the gene of interestmay have high guide strand activity and low passenger strand activity invitro.

In one embodiment, the siRNA molecules have a high guide strand activityand low passenger strand activity in vitro. The target knock-down (KD)by the guide strand may be at least 40%, 50%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 99%, 99.5% or 100%. The target knock-down by the guidestrand may be 40-50%, 45-50%, 50-55%, 50-60%, 60-65%, 60-70%, 60-75%,60-80%, 60-85%, 60-90%, 60-95%, 60-99%, 60-99.5%, 60-100%, 65-70%,65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 65-99%, 65-99,5%, 65-100%,70-75%, 70-80%, 70-85%, 70-90%, 70-95%, 70-99%, 70-99.5%, 70-100%,75-80%, 75-85%, 75-90%, 75-95%, 75-99%, 75-99.5%, 75-100%, 80-85%,80-90%, 80-95%, 80-99%, 80-99.5%, 80-100%, 85-90%, 85-95%, 85-99%,85-99,5%, 85-100%, 90-95%, 90-99%, 90-99.5%, 90-100%, 95-99%, 95-99.5%,95-100%, 99-99.5%, 99-100% or 99.5-100%. As a non-limiting example, thetarget knock-down (KD) by the guide strand is greater than 70%. As anon-limiting example, the target knock-down (KD) by the guide strand isgreater than 60%.

In one embodiment, the highest knock-down from delivery of the siRNAmolecules is seen around the injection site(s).

In one embodiment, knock-down is seen in the ventral horn and around theinjection site(s) after delivery of the siRNA molecules.

In one embodiment, the siRNA duplex is designed so there is no miRNAseed match for the sense or antisense sequence to the non-gene ofinterest sequence.

In one embodiment, the IC₅₀ of the guide strand for the nearest offtarget is greater than 100 multiplied by the IC₅₀ of the guide strandfor the on-target gene. As a non-limiting example, if the IC50 of theguide strand for the nearest off target is greater than 100 multipliedby the IC₅₀ of the guide strand for the target then the siRNA moleculeis said to have high guide strand selectivity for inhibiting the gene ofinterest in vitro.

In one embodiment, the 5′ processing of the guide strand has a correctstart (n) at the 5′ end at least 75%, 80%, 85%, 90%, 95%, 99% or 100% ofthe time in vitro or in vivo, As a non-limiting example, the 5′processing of the guide strand is precise and has a correct start (n) atthe 5′ end at least 99% of the time in vitro. As a non-limiting example,the 5′ processing of the guide strand is precise and has a correct start(n) at the 5′ end at least 99% of the time in vivo, As a non-limitingexample, the 5′ processing of the guide strand is precise and has acorrect start (n) at the 5′ end at least 90% of the time in vitro. As anon-limiting example, the 5′ processing of the guide strand is preciseand has a correct start (n) at the 5′ end at least 90% of the time invivo. As a non-limiting example, the 5′ processing of the guide strandis precise and has a correct start (n) at the 5′ end at least 85% of⁻thetime in vitro. As a non-limiting example, the 5′ processing of the guidestrand is precise and has a correct start (n) at the 5′ end at least 85%of the time in vivo.

In one embodiment, the guide to passenger (G:P) (also referred to as theantisense to sense) strand ratio expressed is 1:10, 1:9, 1:8, 1:7, 1:6,1:5, 1:4, 1:3, 1:2. 1;1, 2:10, 2:9, 2:8, 2:7, 2:6, 2:5, 2:4, 2:3, 2:2,2:1, 3:10, 3:9, 3:8, 3:7, 3:6, 3:5, 3:4, 3:3, 3:2, 3:1, 4:10, 4:9, 4:8,4:7, 4:6, 4:5, 4:4, 4:3, 4:2, 4:1, 5:10, 5:9, 5:8, 5:7, 5:6, 5:5, 5:4,5:3, 5:2, 5:1, 6:10, 6:9, 6:8, 6:7, 6:6, 6:5, 6:4, 6:3, 6:2, 6:1, 7:10,7:9, 7:8, 7:7, 7:6, 7:5, 7:4, 7:3, 7:2, 7:1, 8:10, 8:9, 8:8, 8:7, 8:6,8:5, 8:4, 8:3, 8:2, 8:1, 9:10, 9:9, 9:8, 9:7, 9:6, 9:5, 9:4, 9:3, 9:2,9:1, 10:10, 10:9, 10:8, 10:7, 10:6, 10:5, 10:4, 10:3, 10:2, 10:1, 1:99,5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50,55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, 95:5, or 99:1 invitro or in vivo. The guide to passenger ratio refers to the ratio ofthe guide strands to the passenger strands after intracellularprocessing of the pri-microRNA. For example, an 80:20 guide-to-passengerratio would have 8 guide strands to every 2 passenger strands processedfrom the precursor. As a non-limiting example, the guide-to-passengerstrand ratio is 8:2 in vitro. As a non-limiting example, theguide-to-passenger strand ratio is 8:2 in vivo. As a non-limitingexample, the guide-to-passenger strand ratio is 9:1 in vitro. As anon-limiting example, the guide-to-passenger strand ratio is 9:1 invivo.

In one embodiment, the guide to passenger (G:P) strand ratio is in arange of 1-99, 1.3-99, 5-99, 10-99, 15-99, 20-99, 25-99, 30-99, 35-99,40-99, 45-99, 50-99, 55-99, 60-99, 65-99, 70-99, 75-99, 80-99, 85-99,90-99, 95-99, 1-10, 1-15, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50,1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 5-10, 5-15, 5-20,5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, 5-60, 5-65, 5-70, 5-75, 5-80,5-85, 5-90, 5-95, 10-15, 10-20, 10-25, 10-30, 10-35, 10-40, 10-45,10-50, 10-55, 10-60, 10-65, 10-70, 10-75, 10-80, 10-85, 10-90, 10-95,15-20, 15-25, 15-30, 15-35, 15-40, 15-45, 15-50, 15-55, 15-60, 15-65,15-70, 15-75, 15-80, 15-85, 15-90, 15-95, 20-25, 20-30, 20-35, 20-40,20-45, 20-50, 20-55, 20-60, 20-65, 20-70, 20-75, 20-80, 20-85, 20-90,20-95, 25-30, 25-35, 25-40, 25-45, 25-50, 25-55, 25-60, 25-65, 25-70,25-75, 25-80, 25-85, 25-90, 25-95, 30-35, 30-40, 30-45, 30-50, 30-55,30-60, 30-65, 30-70, 30-75, 30-80, 30-85, 30-90, 30-95, 35-40, 35-45,35-50, 35-55, 35-60, 35-65, 35-70, 35-75, 35-80, 35-85, 35-90, 35-95,40-45, 40-50, 40-55, 40-60, 40-65, 40-70, 40-75, 40-80, 40-85, 40-90,40-95, 45-50, 45-55, 45-60, 45-65, 45-70, 45-75, 45-80, 45-85, 45-90,45-95, 50-55, 50-60, 50-65, 50-70, 50-75, 50-80, 50-85, 50-90, 50-95,55-60, 55-65, 55-70, 55-75, 55-80, 55-85, 55-90, 55-95, 60-65, 60-70,60-75, 60-80, 60-85, 60-90, 60-95, 65-70, 65-75, 65-80, 65-85, 65-90,65-95, 70-75, 70-80, 70-85, 70-90, 70-95, 75-80, 75-85, 75-90, 75-95,80-85, 80-90, 80-95, 85-90, 85-95, or 90-95. As a non-limiting example,the guide to passenger ratio is a range of 1.3 to 99. As a non-limitingexample, the guide to passenger ratio is a range of 10 to 99.

In one embodiment, the guide to passenger (G:P) strand ratio is 10,10,5. 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15,5, 16, 16.5, 17,17,5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24,24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31,31.5, 32, 32.5, 33. 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38,38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44,5, 45,45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52,52.5, 53, 53.5, 54, 54,5, 55, 55.5, 56, 56,5, 57, 57.5, 58, 58.5, 59,59.5, 60, 60.5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65, 65.5, 66,66.5, 67, 67.5, 68, 68.5, 69, 69.5, 70, 70.5, 71, 71.5, 72, 72.5, 73,73.5, 74, 74.5, 75, 75.5, 76, 76.5, 77, 77.5, 78, 78.5, 79, 79.5. 80,80.5, 81. 81.5, 82, 82.5, 83, 83.5, 84, 84.5, 85, 85,5, 86, 86.5, 87,87.5, 88, 88.5, 89, 89.5, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5, 94,94.5, 95. 95.5, 96, 96.5, 97, 97.5, 98, 98.5, or 99. As a non-limitingexample, the guide to passenger (G:P) strand ratio is 11.5. As anon-limiting example, the guide to passenger (G:P) strand ratio is 99.

In one embodiment, the guide to passenger (G:P) (also referred to as theantisense to sense) strand ratio expressed is greater than 1.

In one embodiment, the guide to passenger (G:P) (also referred to as theantisense to sense) strand ratio expressed is greater than 2.

In one embodiment, the guide to passenger (G:P) (also referred to as theantisense to sense) strand ratio expressed is greater than 5.

In one embodiment, the guide to passenger (G:P) (also referred to as theantisense to sense) strand ratio expressed is greater than 10.

In one embodiment, the guide to passenger (G:P) (also referred to as theantisense to sense) strand ratio expressed is greater than 20.

In one embodiment, the guide to passenger (G:P) (also referred to as theantisense to sense) strand ratio expressed is greater than 50.

In one embodiment, the guide to passenger (G:P) (also referred to as theantisense to sense) strand ratio expressed is greater than 300.

In one embodiment, the guide to passenger (G:P) (also referred to as theantisense to sense) strand ratio expressed is 314.

In one embodiment, the guide to passenger (G:P) (also referred to as theantisense to sense) strand ratio expressed is greater than 400.

In one embodiment, the guide to passenger (G:P) (also referred to as theantisense to sense) strand ratio expressed is 434.

In one embodiment, the guide to passenger (G:P) (also referred to as theantisense to sense) strand ratio expressed is at least 3:1.

In one embodiment, the guide to passenger (G:P) (also referred to as theantisense to sense) strand ratio expressed is at least 5:1.

In one embodiment, the guide to passenger (G:P) (also referred to as theantisense to sense) strand ratio expressed is at least 10:1.

In one embodiment, the guide to passenger (G:P) (also referred to as theantisense to sense) and ratio expressed is at least 20:1.

In one embodiment, the guide to passenger (G:P) (also referred to as theantisense to sense) strand ratio expressed is at least 50:1.

In one embodiment, the passenger to guide (P:G) (also referred to as thesense to antisense) strand ratio expressed is 1:10, 1:9, 1:8, 1:7, 1:6,1:5, 1:4, 1:3, 1:2, 1:1, 2:10. 2:9, 2:8, 2:7, 2:6, 2:5, 2:4, 2:3, 2:2,2:1, 3:10, 3:9, 3:8, 3:7, 3:6, 3:5, 3:4, 3:3, 3:2, 3:1, 4:10, 4:9, 4:8,4:7, 4:6, 4:5, 4:4, 4:3, 4:2, 4:1, 5:10, 5:9, 5:8, 5:7, 5:6, 5:5, 5:4,5:3, 5:2, 5:1, 6:10, 6:9, 6:8, 6:7, 6:6, 6:5, 6:4, 6:3, 6:2, 6:1, 7:10,7:9, 7:8, 7:7, 7:6, 7:5, 7:4, 7:3, 7:2, 7:1, 8:10, 8:9, 8:8, 8:7, 8:6,8:5, 8:4, 8:3, 8:2, 8:1, 9:10, 9:9, 9:8, 9:7, 9:6, 9:5, 9:4, 9:3, 9:2,9:1, 10:10, 10:9, 10:8, 10:7, 10:6, 10:5, 10:4, 10:3, 10:2, 10:1, 1:99,5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50,55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, 95:5, or 99:1 invitro or in vivo. The passenger to guide ratio refers to the ratio ofthe passenger strands to the guide strands after the intracellularprocessing of the pri-microRNA. For example, an 80:20 ofpassenger-to-guide ratio would have 8 passenger strands to every 2 guidestrands processed from the precursor. As a non-limiting example, thepassenger-to-guide strand ratio is 80:20 in vitro. As a non-limitingexample, the passenger-to-guide strand ratio is 80:20 in vivo. As anon-limiting example, the passenger-to-guide strand ratio is 8:2 invitro. As a non-limiting example, the passenger-to-guide strand ratio is8:2 in vivo. As a non-limiting example, the passenger-to-guide strandratio is 9:1 in vitro. As a non-limiting example, the passenger-to-guidestrand ratio is 9:1 in vivo.

In one embodiment, the passenger to guide (P:G) strand ratio is in arange of 1-99, 1.3-99, 5-99, 10-99, 15-99, 20-99, 25-99, 30-99, 35-99,40-99, 45-99, 50-99, 55-99, 60-99, 65-99, 70-99, 75-99, 80-99, 85-99,90-99, 95-99, 1-10, 1-15, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50,1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 5-1 5-15, 5-20,5-25. 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, 5-60, 5-65, 5-70, 5-75, 5-80,5-85, 5-905-95, 10-15, 1.0-20, 10-25, 10-30, 10-35, 10-40, 10-45, 10-50,10-55, 10-60, 10-65, 10-70, 10-75, 10-80, 10-85, 10-90, 10-95, 15-20,15-25, 15-30, 15-35, 15-40, 15-45, 15-50, 15-55, 15-60, 15-65, 15-70,15-75, 15-80, 15-85, 15-90, 15-95, 20-25, 2030, 20-35, 20-40, 20-45,20-50, 20-55, 20-60, 20-65, 20-70, 20-75, 20-80, 20-85, 20-90, 20-95,25-30, 25-35, 25-40, 25-45, 25-50, 25-55, 25-60, 25-65, 25-70, 25-75,25-80, 25-85, 25-90, 25-95, 30-35, 30-40, 30-45, 30-50, 30-55, 30-60,30-65, 30-70, 30-75, 30-80, 30-85, 30-90, 30-95, 35-40, 35-45, 35-50,35-55, 35-60, 35-65, 35-70, 35-75, 35-80, 35-85, 35-90, 35-95, 40-45,40-50, 40-55, 40-60, 40-65, 40-70, 40-75, 40-80, 40-85, 40-90, 40-95,45-50, 45-55, 45-60, 45-65, 45-70, 45-75, 45-80, 45-85, 45-90, 45-95,50-55, 50-60, 50-65, 50-70, 50-75, 50-80, 50-85, 50-90, 50-95, 55-60,55-65, 55-70, 55-75, 55-80, 55-85, 55-90, 55-95, 60-65, 60-70, 60-75,60-80, 60-85, 60-90, 60-95, 65-70, 65-75, 65-80, 65-85, 65-90, 65-95,70-75, 70-80, 70-85, 70-90, 70-95, 75-80, 75-85, 75-90, 75-95, 80-85,80-90, 80-95, 85-90, 85-95, or 90-95.

In one embodiment, the passenger to guide (P:G) strand ratio is 10,10.5, 11, 11,5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17,17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24,24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31,31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5. 36, 36.5, 37. 37.5, 38,38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45,45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52,52.5, 53. 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59,59.5, 60, 60.5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64,5, 65, 65.5, 66,665, 67, 67.5, 68, 68.5, 69, 69.5, 70, 70.5, 71, 71.5, 72, 72.5, 73,73.5, 74, 74.5, 75, 75.5, 76, 76.5, 77, 77.5, 78. 78.5, 79, 79.5, 80,80.5, 81, 81.5, 82, 82.5, 83, 83.5, 84, 84.5, 85, 85.5, 86, 86.5, 87,87.5, 88, 88.5, 89, 89.5, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5, 94,94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, or 99.

In one embodiment, the passenger to guide (P:G) (also referred to as thesense o antisense) strand ratio expressed is greater than 1.

In one embodiment, the passenger to guide (P:G) (also referred to as thesense to antisense and ratio expressed is greater than 2.

In one embodiment, the passenger to guide (P:G) (also referred to as thesense to antisense) strand ratio expressed is greater than 5.

In one embodiment, the passenger to guide (P:G) (also referred to as thesense to antisense) strand ratio expressed is greater than 10.

In one embodiment, the passenger to guide (P:G) (also referred to as thesense to antisense) strand ratio expressed is greater than 20.

In one embodiment, the passenger to guide (P:G) (also referred to as thesense to antisense) strand ratio expressed is greater than 50.

In one embodiment, the passenger to guide (P:G) (also referred to as thesense to antisense) strand ratio expressed is at least 3:1.

In one embodiment, the passenger to guide (P:G) (also referred to as thesense to antisense) strand ratio expressed is at least 5:1.

In one embodiment, the passenger to guide (P:G) (also referred to as thesense to antisense) strand ratio expressed is at least 10:1.

In one embodiment, the passenger to guide (P:G) (also referred to as thesense to antisense) strand ratio expressed is at least 20:1.

In one embodiment, the passenger to guide (P:G) (also referred to as thesense to antisense) strand ratio expressed is at least 50:1.

In one embodiment, a passenger-guide strand duplex is consideredeffective when the pri- or pre-microRNAs demonstrate, but methods knownin the art and described herein, greater than 2-fold guide to passengerstrand ratio when processing is measured. As a non-limiting examples,the pri- or pre-microRNAs demonstrate great than 2-fold, 3-fold, 4-fold,5-thld, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold,13-thld, 14-fold, 15-fold, or 2 to 5-fold, 2 to 10-fold, 2 to 15-fold, 3to 5-fold, 3 to 10-fold, 3 to 15-fold, 4 to 5-fold, 4 to 10-fold, 4 to15-fold, 5 to 10-fold, 5 to 1.5-fold, 6 to 10-fold, 6 to 15-fold, 7 to10-fold, 7 to 15-fold, 8 to 10-fold, 8 to 15-fold, 9 to 10-fold, 9 to15-fold, 10 to 15-fold, 11 to 15-fold, 12 to 15-fold, 13 to 15-fold, or14 to 15-fold guide to passenger strand ratio when processing ismeasured.

In one embodiment, the vector genome encoding the dsRNA comprises asequence which is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%or more than 99% of the full length of the construct. As a non-limitingexample, the vector genome comprises a sequence which is at least 80% ofthe full-length sequence of the construct.

In one embodiment, the siRNA molecules may be used to silence wild typeor mutant version of the gene of interest by targeting at least one exonon the gene of interest sequence. The exon may be exon 1, exon 2, exon3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11,exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19,exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27,exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35,exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43,exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51,exon 52, exon 53, exon 54. exon 55, exon 56, exon 57, exon 58, exon 59,exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, and/orexon 67. Design and Sequences of siRNA duplexes targeting SOD1 gene

The present invention provides small interfering RNA (siRNA) duplexes(and modulatory polynucleotides encoding them) that target SOD1 mRNA tointerfere with SOD1 gene expression and/or SOD1 protein production.

The encoded siRNA duplex of the present invention contains an antisensestrand and a sense strand hybridized together forming a duplexstructure, wherein the antisense strand is complementary to the nucleicacid sequence of the targeted SOD1 gene, and wherein the sense strand ishomologous to the nucleic acid sequence of the targeted SOD I gene. Insome aspects, the 5′end of the antisense strand has a 5′ phosphate groupand the 3′end of the sense strand contains a 3′hydroxyl group. In otheraspects, there are none, one or 2 nucleotide overhangs at the 3′end ofeach strand.

Some guidelines for designing siRNAs have been proposed in the art.These guidelines generally recommend generating a 19-nucleotide duplexedregion, symmetric 2-3 nucleotide 3′overhangs, 5′-phosphate and3-hydroxyl groups targeting a region in the gene to be silenced. Otherrules that may govern siRNA sequence preference include, but are notlimited to, (i) A/U at the 5′ end of the antisense strand; (ii) GIC atthe 5′ end of the sense strand; (iii) at least five A/U residues in the5′ terminal one-third of the antisense strand; and (iv) the absence ofany GC stretch of more than 9 nucleotides in length. In accordance withsuch consideration, together with the specific sequence of a targetgene, highly effective siRNA molecules essential for suppressing theSOD1 gene expression may be readily designed.

According to the present disclosure, siRNA molecules (e.g., siRNAduplexes or encoded dsRNA) that target the SOD1 gene are designed. SuchsiRNA molecules can specifically, suppress SOD1 gene expression andprotein production. In some aspects, the siRNA molecules are designedand used to selectively “knock out” SOD1 gene variants in cells, i.e.,mutated SOD1 transcripts that are identified in patients with ALSdisease. In some aspects, the siRNA molecules are designed and used toselectively “knock down” SOD1 gene variants in cells. In other aspects,the siRNA molecules are able to inhibit or suppress both the wild typeand mutated SOD1 gene.

In one embodiment, an siRNA molecule of the present disclosure comprisesa sense strand and a complementary antisense strand in which bothstrands are hybridized together to form a duplex structure. Theantisense strand has sufficient complementarity to the SOD1 mRNAsequence to direct target-specific RNAi, i.e., the siRNA molecule has asequence sufficient to trigger the destruction of the target mRNA by theRNAi machinery or process,

In one embodiment, an siRNA molecule of the present disclosure comprisesa sense strand and a complementary antisense strand in which bothstrands are hybridized together to form a duplex structure and where thestart site of the hybridization to the SODI mRNA is between nucleotide15 and 1000 on the SOD1 mRNA sequence. As a non-limiting example, thestart site may be between nucleotide 15-25, 15-50, 15-75, 15-100,100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500,500-550, 550-600, 600-650, 650-700, 700-70, 750-800, 800-850, 850-900,900-950, and 950-1000 on the SOD1 mRNA sequence. As yet anothernon-limiting example, the start site may be nucleotide 26, 27, 28, 29,30, 32, 33, 34, 35, 36, 37, 74, 76, 77, 78, 149, 153, 157, 160, 177,192, 193, 195, 196, 197, 198, 199. 206, 209, 210, 239, 241, 261, 263.264, 268, 269, 276, 278, 281, 284, 290, 291, 295, 296, 316, 317, 329,330, 337, 350, 351, 352, 354, 357, 358, 364, 375, 378, 383, 384, 390,392, 395, 404, 406, 417, 418, 469, 470, 475, 476, 480, 487, 494, 496,497, 501, 504, 515. 518, 522, 523, 524, 552, 554, 555, 562, 576, 577,578, 579, 581, 583, 584, 585, 587, 588. 589, 593, 594, 595, 596, 597,598, 599, 602. 607, 608, 609, 610, 611, 612, 613, 616, 621, 633, 635,636, 639, 640, 641, 642, 643, 644, 645, 654, 660, 661, 666, 667, 668,669, 673, 677, 692, 698, 699, 700, 701, 706, 749, 770, 772, 775, 781,800, 804, 819, 829, 832, 833, 851. 854, 855, 857, 858, 859, 861, 869.891, 892, 906, 907, 912, 913, 934, 944, and 947 on the SOD1 mRNAsequence.

In some embodiments, the antisense strand and target SOD1 mRNA sequenceshave 100% complementarity. The antisense strand may be complementary toany part of the target SOD1 mRNA sequence.

In other embodiments, the antisense strand and target SODI. niRNAsequences comprise at least one mismatch. As a non-limiting example, theantisense strand and the target SOD1 mRNA sequence have at least 30%,40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or at least 20-30%,20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-99%, 30-40%,30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-99%, 40-50%, 40-60%,40-70%, 40-80%, 40-90%, 40-95%, 40-99%, 50-60%, 50-70%, 50-80%, 50-90%,50-95%, 50-99%, 60-70%, 60-80%, 60-90%, 60-95%, 60-99%, 70-80%, 70-90%,70-95%, 70-99%, 80-90%, 80-95%, 80-99%, 90-95%, 90-99% or 95-99%complementarity.

In one embodiment, an siRNA or dsRNA targeting SOD1 includes at leasttwo sequences that are complementary to each other.

According to the present disclosure, the siRNA molecule targeting SOD1has a length from about 10-50 or more nucleotides, i.e., each strandcomprising 10-50 nucleotides (or nucleotide analogs). Preferably, thesiRNA molecule has a length from about 15-30, e.g., 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in eachstrand, wherein one of the strands is sufficiently complementarity to atarget region. In one embodiment, each strand of the siRNA molecule hasa length from about 19 to 25, 19 to 24 or 19 to 21 nucleotides, In oneembodiment, at least one strand of the siRNA molecule is 19 nucleotidesin length. In one embodiment, at least one strand of the siRNA moleculeis 20 nucleotides in length. In one embodiment, at least one strand ofthe siRNA molecule is 21 nucleotides in length. In one embodiment, atleast one strand of the siRNA. molecule is 22 nucleotides in length. Inone embodiment, at least one strand of the siRNA molecule is 23nucleotides in length. In one embodiment, at least one strand of thesiRNA molecule is 24 nucleotides in length. In one embodiment, at leastone strand of the siRNA molecule is 25 nucleotides in length.

In some embodiments, the siRNA molecules of the present disclosuretargeting SOD1 can be synthetic RNA duplexes comprising about 19nucleotides to about 25 nucleotides, and two overhanging nucleotides atthe 3′-end. In some aspects, the siRNA molecules may be unmodified RNAmolecules. In other aspects, the siRNA molecules may contain at leastone modified nucleotide, such as base, sugar or backbone modifications.

In one embodiment, the siRNA molecules of the present disclosuretargeting SOD1 may comprise a nucleotide sequence such as, but notlimited to, the antisense (guide) sequences in Table 2 or a fragment orvariant thereof As a non-limiting example, the antisense sequence usedin the siRNA molecule of the present disclosure is at least 30%, 40%,50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or at least 20-30%,20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90, 20-95%, 20-99%, 30-40%,30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-99%, 40-50%, 40-60%,40-70%, 40-80%, 40-90%, 40-95%, 40-99%, 50-60%, 50-70%, 50-80%, 50-90%,50-95%, 50-99%, 60-70%, 60-80%, 60-90%, 60-95%, 60-99%, 70-80%, 70-90%,70-95%, 70-99%, 80-90%, 80-95%, 80-99%, 90-95%, 90-99% or 95-99% of anucleotide sequence in Table 2. As another non-limiting example, theantisense sequence used in the siRNA molecule of the present disclosurecomprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. 15, 10, 17,18, 19, 20, 21 or more than 21 consecutive nucleotides of a nucleotidesequence in Table 2. As yet another non-limiting example, the antisensesequence used in the siRNA molecule of the present disclosure comprisesnucleotides 1 to 22, 1 to 21, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to8, 2 to 22, 2 to 21, 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to15, 2 to 14, 2 to 13, 2 to 12, 2 to 2 to 10, 2 to 9, 2 to 8, 3 to 22, 3to 21, 3 to 20, 3 to 19, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 4 to 22, 4 to 21, 4 to20, 4 to 19, 4 to 18, 4 to 17, 4 to 16, 4 to 15, 4 to 14, 4 to 13, 4 to12, 4 to 11,4 to 10, 4 to 9, 4 to 8, 5 to 22, 5 to 21, 5 to 20, 5 to 19,5 to 18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11,5 to 10, 5 to 9, 5 to 8, 6 to 22, 6 to 21, 6 to 20, 6 to 19, 6 to 18, 6to 17, 6 to 16, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 7to 22, 7 to 21, 7 to 20, 7 to 19, 7 to 18, 7 to 17, 7 to 16, 7 to 15, 7to 14, 7 to 13, 7 to 12, 8 to 22, 8 to 21, 8 to 20, 8 to 19, 8 to 18, 8to 17, 8 to 16, 8 to 15, 8 to 14, 8 to 13, 8 to 12, 9 to 22, 9 to 21, 9to 20, 9 to 19, 9 to 18, 9 to 17, 9 to 16, 9 to 15, 9 to 14, 10 to 22,10 to 21, 10 to 20, 10 to 19, 10 to 18, 10 to 17, 10 to 16, 10 to 15, 10to 14, 11 to 22, 11 to 21, 11 to 20, 11 to 19, 11 to 18, 11 to 17, 11 to16, 11 to 15, 11 to 14, 12 to 22, 12 to 21, 12 to 20, 12 to 19, 12 to18, 12 to 17, 12 to 16, 13 to 22, 13 to 21, 13 to 20, 13 to 19, 13 to18, 13 to 17, 13 to 16, 14 to 22, 14 to 21, 14 to 20, 14 to 19, 14 to18, 14 to 17, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 16 to22, 16 to 21, 16 to 20, 17 to 22, 17 to 21, or 18 to 22 of the sequencesin Table 2.

TABLE 2  Antisense Sequences Antisense ID Sequence SEQ ID NO A-4002UAUUAAAGUGAGGACCUGCUU 1

In one embodiment, the siRNA molecules of the present disclosuretargeting SOD1 may comprise a nucleotide sequence such as, but notlimited to, the sense (passenger) sequences in Table 3 or a fragment orvariant thereof, As a non-limiting example, the sense sequence used inthe siRNA molecule of the present disclosure is at least 30%, 40%, 50%,60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or at least 20-30%, 20-40%,20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-99%, 30-40%, 30-50%,30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-99%, 40-50%, 40-60%, 40-70%,40-80%, 40-90%, 40-95%, 40-99%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%,50-99%, 60-70%, 60-80%, 60-90%, 60-95%, 60-99%, 70-80%, 70-90%, 70-95%,70-99%, 80-90%, 80-95%, 80-99%, 90-95%, 90-99% or 95-99% of a nucleotidesequence in Table 3. As another non-limiting example, the sense sequenceused in the siRNA molecule of the present disclosure comprises at least3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 ormore than 21 consecutive nucleotides of a nucleotide sequence in Table3. As yet another non-limiting example, the sense sequence used in thesiRNA molecule of the present disclosure comprises nucleotides 1 to 22,1 to 21, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14,1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 2 to 22, 2 to 21, 2to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2to 1.2, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 3 to 22, 3 to 21, 3 to 20, 3to 19, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3to 11, 3 to 10, 3 to 9, 3 to 8, 4 to 22, 4 to 21, 4 to 20, 4 to 19, 4 to18, 4 to 17, 4 to 16, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to10, 4 to 9, 4 to 8, 5 to 22, 5 to 21, 5 to 20, 5 to 19, 5 to 18, 5 to17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to9, 5 to 8, 6 to 22, 6 to 21, 6 to 20, 6 to 19, 6 to 18, 6 to 17, 6 to16, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 7 to 22, 7 to21, 7 to 20, 7 to 19, 7 to 18, 7 to 17, 7 to 16, 7 to 15, 7 to 14, 7 to13, 7 to 12, 8 to 22, 8 to 21, 8 to 20, 8 to 19, 8 to 188 to 17, 8 to16, 8 to 15, 8 to 14, 8 to 13, 8 to 12, 9 to 22, 9 to 21, 9 to 20, 9 to19, 9 to 18, 9 to 17, 9 to 16, 9 to 15, 9 to 14, 10 to 22, 10 to 21, 10to 20, 10 to 19, 10 to 18, 10 to 17, 10 to 16, 10 to 15, 10 to 14, 11 to22, 11 to 21, 11 to 20, 11 to 19, 11 to 18, 11 to 17, 11 to 16, 11 to15, 11 to 14, 12 to 22, 12 to 21, 12 to 20, 12 to 19, 12 to 18, 12 to1712 to 16, 13 to 22, 13 to 21, 13 to 20, 13 to 9, 13 to 18, 13 to 17,13 to 16, 14 to 22, 14 to 21, 14 to 20, 14 to 19, 14 to 18, 14 to 17, 15to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 16 to 22, 16 to 21, 16 to20, 17 to 22, 17 to 21, or 18 to 22 of the sequences in Table 3.

TABLE 3  Sense Sequences Sense ID Sequence SEQ ID NO S-4003GCAGGUCCUCACUUUAAUGCU 2

In one embodiment, the siRNA molecules of the present disclosuretargeting SOD1 may comprise an antisense sequence from Table 2 and asense sequence from Table 3, or a fragment or variant thereof. As anon-limiting example, the antisense sequence and the sense sequence haveat least 30%, 40%, 500o, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% or atleast 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%,20-99%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-99%,40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-99%, 50-60%, 50-70%,50-80%, 50-90%, 50-95%, 50-99%, 60-70%, 60-80%, 60-90%, 60-95%, 60-99%70-80%, 70-90%, 70-95%, 70-99%, 80-90%, 80-95%, 80-99%, 90-95%, 90-99%or 95-99% complementarity.

In one embodiment, the siRNA molecules of the present disclosuretargeting SOD1 may comprise the sense and antisense siRNA duplex asdescribed in Table 4. As a non-miting example, these siRNA duplexes maybe tested for in vitro inhibitory activity on endogenous SOD1 geneexpression.

TABLE 4  Sense and antisense strand sequences of SOD1 dsRNA AntisensesiRNA Sense Strand SS Strand AS Duplex Sequence SEQ Sequence SEQ IDSS ID (5′-3′) ID AS ID (5′-3′) ID D-4012 S-4003 GCAGGUCCUCA 2 A-4002UAUUAAAGUGA 1 CUUUAAUGCU GGACCUGCUU

In other embodiments, the siRNA molecules of the present disclosuretargeting SOD1 can be encoded in plasmid vectors, AAV particles, viralgenome or other nucleic acid expression vectors for delivery to a cell.

DNA expression plasmids can be used to stably express the siRNA duplexesor dsRNA of the present disclosure targeting SOD1 in cells and achievelong-term inhibition of the target gene expression. In one aspect, thesense and antisense strands of a siRNA duplex are typically linked by ashort spacer sequence leading to the expression of a stem-loop structuretermed short hairpin RNA (shRNA), The hairpin is recognized and cleavedby Dicer, thus generating mature siRNA molecules.

According to the present disclosure, AAV particles comprising thenucleic acids encoding the siRNA molecules targeting SOD1 mRNA areproduced, the AAV serotypes may be any of the serotypes listed herein.Non-limiting examples of the AAV serotypes include, AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14) AAV10, AAV11,AAV12, AAVrh8, AAVrh10, AAV-DJ, AAV-PHRA, and/or AAV-PHP.B, AAVPHP.B2,AAVPHP.B3, AAVPHP.N/PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP,AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS,AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT,AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST,AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQPNAVPHP.B-QLP,AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S/G2A12, AAVG2A15/G2A3, AAVG2B4,AAVG2R5, and variants thereof.

In some embodiments, the siRNA duplexes or encoded dsRNA of the presentdisclosure suppress (or degrade) SOD1 mRNA. Accordingly, the siRNAduplexes or encoded dsRNA can be used to substantially inhibit SOD1 geneexpression in a cell. In some aspects, the inhibition of SOD1 geneexpression refers to an inhibition by at least about 20%, preferably byat least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, orat least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%,20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%,30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%,50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%,60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%,80-100%, 90-95%, 90-100% or 95-100%. Accordingly, the protein productof⁻the targeted gene may be inhibited by at least about 20%, preferablyby at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%,or at least 20-30%. 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%,20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%,30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%,50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%,60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%,80-100%, 90-95%, 90-100% or 95-100%.

According to the present disclosure, the siRNA molecules are designedand tested for their ability in reducing SOD1 mRNA levels in culturedcells. Such siRNA molecules may form a duplex such as, but not limitedto, include those listed in Table 4. As a non-limiting example, thesiRNA duplexes may be siRNA duplex ID D-4012,

In one embodiment, the siRNA molecules comprise a miRNA seed match forSOD1 located in the guide strand, In another embodiment, the siRNAmolecules comprise a miRNA seed match for SOD1 located in the passengerstrand. In yet another embodiment, the siRNA duplexes or encoded dsRNAtargeting SOD1 gene do not comprise a seed match for SOD1 located in theguide or passenger strand.

In one embodiment, the siRNA duplexes or encoded dsRNA targeting SOD1gene may have almost no significant full-length off target effects forthe guide strand. In another embodiment, the siRNA. duplexes or encodeddsRNA targeting SOD1 gene may have almost no significant full-length offtarget effects for the passenger strand. The siRNA duplexes or encodeddsRNA targeting SOD1 gene may have less than 1%, 2%, 3%, 4%, 5%, 6%, 7%.8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,1-5%, 2-6%, 3-7%, 4-8%, 5-9%, 5-10%, 6-10%, 5-15%, 5-20%, 5-25% 5-30%,10-20%, 10-30%, 10-40%, 10-50%, 15-30%, 15-40%, 15-45%, 20-40%, 20-50%,25-50%, 30-40%, 30-50%, 35-50%, 40-50%, 45-50% full-length off targeteffects for the passenger strand. In yet another embodiment, the siRNAduplexes or encoded dsRNA targeting SOD1 gene may have almost nosignificant full-length off target effects for the guide strand or thepassenger strand. The siRNA duplexes or encoded dsRNA targeting SOD1gene may have less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 1-5%, 2-6%, 3-7%,4-8%, 5-9%, 5-10%, 6-10%, 5-15%, 5-20%, 5-25% 5-30%, 10-20%, 10-30%,10-40%, 10-50%, 15-30%, 15-40%, 15-45%, 20-40%, 20-50%, 25-50%, 30-40%,30-50%, 35-50%, 40-50%, 45-50% full-length off target effects for theguide or passenger strand.

In one embodiment, the siRNA duplexes or encoded dsRNA targeting SOD1gene may have high activity in vitro. In another embodiment, the siRNAmolecules may have low activity in vitro. In yet another embodiment, thesiRNA duplexes or dsRNA targeting the SOD1 gene may have high guidestrand activity and low passenger strand activity in vitro.

In one embodiment, the siRNA molecules targeting SOD1 have a high guidestrand activity and low passenger strand activity in vitro. The targetknock-down (KD) by the guide strand may be at least 40%, 50%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5% or 100%, The target knock-downby the guide strand may be 40-50%, 45-50%, 50-55%, 50-60%, 60-65%,60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 60-99%, 60-99.5%,60-100%, 65-70%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 65-99%,65-99.5%, 65-100%, 70-75%, 70-80%, 70-85%, 70-90%, 70-95%, 70-99%,70-99.5%, 70-100%, 75-80%, 75-85%, 75-90%, 75-95%, 75-99%, 75-99.5%,75-100%, 80-85%, 80-90%, 80-95%, 80-99%, 80-99.5%, 80-100%, 85-90%,85-95%, 85-99%, 85-99.5%, 85-100%, 90-95%, 90-99%, 90-99.5%, 90-100%,95-99%, 95-99.5%, 95-100%, 99-99.5%, 99-100% or 99.5-100%. As anon-limiting example, the target knock-down (KD) by the guide strand isgreater than 70%. As a non-limiting example, the target knock-down (KD)by the guide strand is greater than 60%.

In one embodiment, the siRNA duplex target SOD1 is designed so there isno miRNA seed match for the sense or antisense sequence to the non-SOD1sequence.

In one embodiment, the IC₅₀ of the guide strand in the siRNA duplextargeting SOD1 for the nearest off target is greater than 100 multipliedby the IC₅₀ of the guide strand for the on-target gene, SOD1. As anon-limiting example, if the IC₅₀ of the guide strand for the nearestoff target is greater than 100 multiplied by the IC₅₀ of the guidestrand for the target then the siRNA molecules are said to have highguide strand selectivity for inhibiting SOD1 in vitro.

In one embodiment, the 5′ processing of the guide strand of the siRNAduplex targeting SOD1 has a correct start (n) at the 5′ end at least75%, 80%, 85%, 90%, 95%, 99% or 100% of the time in vitro or in vivo. Asa non-limiting example, the 5′ processing of the guide strand is preciseand has a correct start (n) at the 5′ end at least 99% of the time invitro. As a non-limiting example, the 5′ processing of the guide strandis precise and has a correct start (n) at the 5′ end at least 99% of thetime in vivo. As a non-limiting example, the 5′ processing of the guidestrand is precise and has a correct start (n) at the 5′ end at least 90%of the time in vitro. As a non-limiting example, the 5′ processing ofthe guide strand is precise and has a correct start (n) at the 5′ end atleast 90% of the time in vivo. As a non-limiting example, the 5′processing of the guide strand is precise and has a correct start (n) atthe 5′ end at least 85% of the time in vitro. As a non-limiting example,the 5′ processing of the guide strand is precise and has a correct start(n) at the 5′ end at least 85% of the time in vivo.

In one embodiment, the 5′ processing of the guide strand of the siRNAduplex targeting SOD1 has a correct start (n) at the 5′ end in a rangeof 75-95%, 75-90%, 75-85%, 75-80%, 80-95%, 80-90%, 80-85%, 85-95%,85-90%, or 90-95%. As a non-limiting example, the 5′ processing of theguide strand of the siRNA duplex targeting SOD1 has a correct start (n)at the 5′ end in a range of 75-95%.

In one embodiment, the 5′ processing of the guide strand of the siRNAduplex targeting SOD1 has a correct start (n) at the 5′ end for 75%,75.1%, 75.2%, 75.3%, 75.4%, 75.5%, 75.6%, 75.7%, 75.8%, 75.9%, 76%,76.1%, 76.2%, 76.3%, 76.4%, 76.5%, 76.6%, 76.7%, 76.8%, 76.9%, 77%,77.1%, 77.2%, 77.3%, 77.4%, 77.5%, 77.6%, 77.7%, 77.8%, 77.9%, 78%,78.1%, 78.2%, 78.3%, 78.4%, 78.5%, 78.6%, 78.7%, 78.8%, 79%, 79.1%,79.7%, 79.3%, 79.4%, 79.5%, 79.6%, 79.7%, 79.8%, 79.9%, 80%, 80.1%,80.2%, 80.3%, 80.4%, 80.5%, 80.6%, 80.7%, 80.8%, 80.9%, 8.10%, 81.1%,81.2%, 81.3%, 81.4%, 81.5%, 81.6%, 81.7%, 81.8%, 81.9%, 82%, 82.1%,82.2%, 82.3%, 82.4%, 82.5%, 82.6%, 82.7%, 82.8%, 82.9%, 83%, 83.1%,83.2%, 83.3%, 83.4%, 83.5%, 83.6%, 83.7%, 83.8%, 83.9%, 84%, 84.1%,84.2%, 84.3%, 84.4%, 84.5%. 84.6%, 84.7%, 84.8%, 84.9%, 85%, 85.1%,85.2%, 85.3%, 85.4%, 85,5%, 85.6%, 85.7%, 85.8%, 85.9%, 86%, 86.1%,86.2%. 86.3%, 86.4%, 86.5%, 86.6%, 86.7%, 86.8%, 86.9%, 87%, 87.1%,87.2%, 87.3%, 87.4%, 87.5%, 87.6%, 87.7%, 87.8%, 87.9%, 88%, 88.1%,88.2%, 88.3%, 88.4%, 88.5%, 88.6%, 88.7%, 88.8%, 88.9%, 89%, 89.1%,89.2%, 89.3%, 89.4%, 89.5%, 89.6%, 89.7%, 89.8%, 89,9%, 90%, 90.1%,90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90,7%, 90.8%, 90.9%, 91%, 91.1%,91.2%, 91.3%, 91.4%, 91.5%, 91.6%, 91.7%, 91.8%, 91.9%, 92%, 92.1%,92.2%, 92.3%, 92.4%, 92.5%, 92.6%, 92.7%, 92.8%, 92.9%, 93%, 93.1%,93.2%, 93.3%, 93.4%, 93.50 , 93.6%. 93.7%, 93.8%, 93.9%, 94%, 94.1%,94.2%, 94.3%, 94.4%, 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, or 95% of theconstructs expressed. As a non-limiting example, the 5′ processing ofthe guide strand of the siRNA duplex targeting SOD1 has a correct start(n) at the 5′ end for 81% of the constructs expressed. As a non-limitingexample. the 5′ processing of the guide strand of the siRNA duplextargeting SOD1 has a correct start (n) at the 5′ end for 90% of theconstricts expressed.

In one embodiment, a passenger-guide strand duplex for SOD1 isconsidered effective when the pri- or pre-microRNAs demonstrate, bymethods known in the art and described herein, greater than 2-fold guideto passenger strand ratio when processing is measured. As a non-limitingexamples, the pri- or pre-microRNAs demonstrate great than 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,11-fold, 12-fold, 13-fold, 14-fold, 15-fold, or 2 to 5-fold, 2 to10-fold, 2 to 15-fold, 3 to 5-fold, 3 to 10-fold, 3 to 15-fold, 4 to5-fold, 4 to 10-fold, 4 to 15-fold, 5 to 10-fold, 5 to 15-fold, 6 to10-fold, 6 to 15-fold, 7 to 10-fold, 7 to 15-fold, 8 to 10-fold, 8 to15-fold, 9 to 10-fold, 9 to 15-fold, 10 to 15-fold, 11 to 15-fold, 12 to15-fold, 13 to 15-fold, or 14 to 15-fold guide to passenger strand ratiowhen processing is measured.

In one embodiment, the siRNA molecules may be used to silence wild typeor mutant SOD1 by targeting at least one exon on the SOD1 sequence. Theexon may be exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon16, exon 17, exon 18, exon 19, exon 20, exon 21, exon exon 23, exon 24,exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32,exon 33, exon 34, exon 35, exon 36, exon 37, exon 38. exon 39, exon 40,exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48.exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56,exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64,exon 65, exon 66, and/or exon 67.

In one embodiment, the range of guide strands to the total endogenouspool of miRNAs is 0.001-0.6%, 0.005-0.6%, 0.01-0.6%, 0.015-0.6%,0,02-0.6%, 0.025-0.6%, 0.03-0.6%, 0.035-0.6%, 0.04-0.6%, 0.045-0.6%,0.05-0.6%, 0.055-0.6%, 0.06-0.6%, 0.065-0.6%, 0.07-0.6%, 0.075-0.6%,0.08-0.6%, 0.085-0.6%, 0.09-0.6%, 0.095-0.6%, 0.1-0.6%, 0.15-0.6%,0.2-0.6%, 0.25-0.6%, 0.3-0.6%, 0.35-0.6%, 0.4-0.6%, 0.45-0.6%, 0.5-0.6%,0.55-0.6%, 0.001-0.5%, 0.005-0.5%, 0.01-0.5%, 0.015-0.5%, 0.02-0.5%,0.025-0.5%, 0.03-0.5%, 0.035-0.5%, 0.04-0.5%, 0.045-0.5%, 0.05-0.5%,0.055-0.5%, 0.06-0.5%, 0.065-0.5%. 0.07-0.5%, 0.075-0.5%, 0.08-0.5%,0.085-0.5%, 0.09-0.5%, 0.095-0.5%, 0.1-0.5%, 0.15-0.5%, 0.2-0.5%,0.25-0.5%, 0.3-0.5%, 0.35-0.5%, 0.4-0.5%, 0.45-0.5%, 0.001-0.4%,0.005-0.4%, 0.01-0.4%, 0.015-0.4%, 0.02-0.4%, 0.025-0.4%, 0.03-0.4%,0.035-0.4%, 0.04-0.4%, 0.045-0.4%, 0.05-0.4%, 0.055-0.4%, 0.06-0.4%,0.065-0.4%, 0.07-0.4%, 0.075-0.4%, 0.08-0.4%, 0.085-0.4%, 0.09-0.4%,0.095-0.4%, 0.1-0.4%, 0.15-0.4%, 0.2-0.4%, 0.25-0.4%, 0.3-0.4%,0.35-0.4%, 0.001-0.3%, 0.005-0.3%, 0.01-0.3%, 0.015-0.3%, 0.02-0.3%,0.025-0.3%, 0.03-0.3%, 0.035-0.3%, 0.04-0.3%, 0.045-0.3%, 0.05-0.3%,0.055-0.3%, 0.06-0.3%, 0.065-0.3%, 0.07-0.3%, 0.075-0.3%, 0.08-0.3%,0.085-0.3%, 0.09-0.3%, 0.095-0.3%, 0.1-0.3%, 0.15-0.3%, 0.2-0.3%,0.25-0.3%, 0.001-0.2%, 0.005-0.2%, 0.01-0.2%, 0.015-0.2%, 0.02-0.2%,0.025-0.2%, 0.03-0.2%, 0.035-0.2%, 0.04-0.2%, 0.045-0.2%, 0.05-0.2%,0.055-0.2%, 0.06-0.2%, 0.065-0.2%, 0.07-0.2%, 0.075-0.2%, 0.08-0.2%,0.085-0.2%, 0.09-0.2%, 0.095-0.2%, 0.1-0.2%, 0.15-0.2%, 0.001-0.1%,0.005-0.1%, 0.01-0.1%, 0.015-0.1%, 0.02-0.1%, 0.025 -0.1%, 0.03-0.1%,0.035-0.1%, 0.04-0.1%, 0.045-0.1%, 0.05-0.1%, 0.055-0.1%, 0.06-0.1%,0.065-0.1%, 0.07-0.1%, 0.075-0.1%, 0.08-0.1%, 0.085-0.1%, 0.09-0.1%, or0.095-0.1%. As a non-limiting example, the range is 0.06-0.6%. As anon-limiting example, the range is 0.4-0.5%.

In one embodiment, the percent of guide strands to the total endogenouspool of miRNAs is 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%,0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%,0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, or 0.6%. As a non-limitingexample, the percent is 0.06%. As a non-limiting example, the percent is0.4%. As a non-limiting example, the percent is 0.5%.

siRNA modification

In some embodiments, the siRNA molecules of the present disclosure, whennot delivered as a precursor or DNA, may be chemically modified tomodulate some features of RNA molecules, such as, but not limited to,increasing the stability of siRNAs in vivo. The chemically modifiedsiRNA molecules can be used in human therapeutic applications, and areimproved without compromising the RNAi activity of the siRNA molecules.As a non-limiting example, the siRNA molecules modified at both the 3′and the 5′ end of both the sense strand and the antisense strand.

In some aspects, the siRNA duplexes of the present disclosure maycontain one or more modified nucleotides such as, but not limited to,sugar modified nucleotides, nucleobase modifications and/or backbonemodifications. In some aspects, the siRNA molecule may contain combinedmodifications, for example, combined nucleobase and backbonemodifications.

In one embodiment, the modified nucleotide may be a sugar-modifiednucleotide. Sugar modified nucleotides include, but are not limited to2′-fluoro, 2′-amino and 2′-thio modified ribonucleotides, e.g. 2′-fluoromodified ribonucleotides. Modified nucleotides may be modified on thesugar moiety, as well as nucleotides having sugars or analogs thereofthat are not ribosyl. For example, the sugar moieties may be, or bebased on, mannoses, arabinoses, glucopyranoses, galactopyranoses,4′-thioribose, and other sugars, heterocycles, or carbocycles.

In one embodiment, the modified nucleotide may be a nucleobase-modifiednucleotide.

In one embodiment, the modified nucleotide may be a backbone-modifiednucleotide. In some embodiments, the siRNA. duplexes of the presentdisclosure may further comprise other modifications on the backbone. Anormal “backbone”, as used herein, refers to the repeating alternatingsugar-phosphate sequences in a DNA or RNA molecule. Thedeoxyriboselribose sugars are joined at both the 3′-hydroxyl and5′-hydroxyl groups to phosphate groups in ester links, also known as“phosphodiester” bonds/linker (PO linkage) The PO backbones may bemodified as “phosphorothioate backbone (PS linkage). In some cases, thenatural phosphodiester bonds may be replaced by amide bonds but the fouratoms between two sugar units are kept. Such amide modifications canfacilitate the solid phase synthesis of oligonucleotides and increasethe thermodynamic stability of a duplex formed with siRNA complement.See e.g. Mesmaeker et al., Pure & Appl. Chem., 1997, 3, 437-440; thecontent of which is incorporated herein by reference in its entirety.

Modified bases refer to nucleotide bases such as, for example, adenine,guanine, cytosine, thymine, uracil, xanthine, inosine, and queuosinethat have been modified by the replacement or addition of one or moreatoms or groups. Some examples of modifications on the nucleobasemoieties include, but are not limited to, alkylated, halogenated,thiolated, aminated, amidated, or acetylated bases, individually or incombination. More specific examples include, for example,5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine,N,N,-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine,1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine andother nucleotides having a modification at the 5 position,5-(2-amino)propyl uridine, 5-halocytidine, 5-halouridine,4-acetylcytidine, 1-methyladenosine, 2-methyladenosine,3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine,2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine,deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine,6-azothymidine, 5-methyl-2-thiouridine, other thio bases such as2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine,pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthylgroups, any O- and N-alkylated purines and. pyrimidines such asN6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyaceticacid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groupssuch as aminophenol or 2,4,6-trimethoxy benzene, modified cytosines thatact as G-clamp nucleotides, 8-substituted adenines and guanines,5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkvlnucleotides, carboxyalkylaminoalkyl nucleotides, andalkylcarbonylalkylated nucleotides.

In one embodiment, the modified nucleotides may be on just the sensestrand.

In another embodiment, the modified nucleotides may be on just theantisense strand.

In some embodiments, the modified nucleotides may be in both the senseand antisense strands.

In some embodiments, the chemically modified nucleotide does not affectthe ability of the antisense strand to pair with the target inRNAsequence.

In one embodiment, the AAV particle comprising a nucleic acid sequenceencoding the siRNA molecules of the present disclosure may encode siRNAmolecules which are polycistronic molecules. The siRNA molecules mayadditionally comprise one or more linkers between regions of the siRNAmolecules.

Molecular Scaffold

In one embodiment, the siRNA molecules may be encoded in a modulatorypolynucleotide which also comprises a molecular scaffold. As used hereina “molecular scaffold” is a framework or starting molecule that formsthe sequence or structural basis against which to design or make asubsequent molecule.

In one embodiment, the molecular scaffold comprises at least one 5′flanking region, As a non-limiting example, the 5′ flanking region maycomprise a 5′ flanking sequence which may be of any length and may bederived in whole or in part from wild type microRNA sequence or be acompletely artificial sequence.

In one embodiment, the molecular scaffold comprises at least one 3′flanking region. As a non-limiting example, the 3′ flanking region maycomprise a 3′ flanking sequence which may be of any length and may bederived in whole or in part from wild type microRNA sequence or be acompletely artificial sequence.

In some embodiments, one or both of the 5′ and 3′ flanking sequences areabsent.

In some embodiments the 5′ and 3′ flanking sequences are the samelength.

In some embodiments the 5′ flanking sequence is from 1-10 nucleotides inlength, from 5-15 nucleotides in length, from 10-30 nucleotides inlength, from 20-50 nucleotides in length, greater than 40 nucleotides inlength, greater than 50 nucleotides in length, greater than 100nucleotides in length or greater than 200 nucleotides in length.

In some embodiments, the 5′ flanking sequence may be 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13. 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308,309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322,323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336,337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350,351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364,365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378,379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392,393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406,407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420,421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434,435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448,449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462,463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476,477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490,491_492, 493, 494, 495, 496, 497, 498, 499, or 500 nucleotides inlength.

In some embodiments the 3′ flanking sequence is from 1-10 nucleotides inlength, from 5-15 nucleotides in length, from 10-30 nucleotides inlength, from 20-50 nucleotides in length, greater than 40 nucleotides inlength, greater than 50 nucleotides in length, greater than 100nucleotides in length or greater than 200 nucleotides in length.

In some embodiments, the 3′ flanking sequence may be 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11 12 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308,309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322,323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336,337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350,351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364,365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378,379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392,393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406,407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420,421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434,435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448,449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462,463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476,477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490,491_492, 493, 494, 495, 496, 497, 498, 499, or 500 nucleotides inlength.

In some embodiments the 5′ and 3′ flanking sequences are the samesequence. In some embodiments they differ by 2%, 3%, 4%, 5%, 10%, 20% ormore than 30% when aligned to each other.

In one embodiment, the molecular scaffold comprises at least one loopmotif region. As a non-limiting example, the loop motif region maycomprise a sequence which may be of any length.

In one embodiment, the molecular scaffold comprises a 5′ flankingregion, a loop motif region and/or a 3′ flanking region.

In one embodiment, at least one siRNA, miRNA or other RNAi agentdescribed herein, may be encoded by a modulatory polynucleotide whichmay also comprise at least one molecular scaffold. The molecularscaffold may comprise a 5′ flanking sequence which may be of any lengthand may be derived in whole or in part from wild type microRNA sequenceor be completely artificial. The 3′ flanking sequence may mirror the 5′flanking sequence and/or a 3′ flanking sequence in size and origin.Either flanking sequence may be absent. The 3′ flanking sequence mayoptionally contain one or more CNNC motifs, where “N” represents anynucleotide.

Forming the stem of a stem loop structure is a minimum of the modulatorypolynucleotide encoding at least one siRNA, miRNA or other RNAi agentdescribed herein. In some embodiments, the siRNA, miRNA or other RNAiagent described herein comprises at least one nucleic acid sequencewhich is in part complementary or will hybridize to a target sequence.In some embodiments the payload is an siRNA molecule or fragment of ansiRNA molecule.

In some embodiments, the 5′ arm of the stein loop structure of themodulatory polynucleotide comprises a nucleic acid sequence encoding asense sequence. Non-limiting examples of sense sequences, or fragmentsor variants thereof, which may be encoded by the modulatorypolynucleotide are described in Table 3.

In some embodiments, the 3′ arm of the stem loop of the modulatorypolynucleotide comprises a nucleic acid sequence encoding an antisensesequence. The antisense sequence, in some instances, comprises a “G”nucleotide at the 5′ most end. Non-limiting examples of antisensesequences, or fragments or variants thereof, which may be encoded by themodulatory polynucleotide are described in Table 2.

In other embodiments, the sense sequence may reside on the 3′ arm whilethe antisense sequence resides on the 5′ arm of the stem of the stemloop structure of the modulatory polynucleotide. Non-limiting examplesof sense and antisense sequences which may be encoded by the modulatorypolynucleotide are described in Tables 2 and 3.

In one embodiment, the sense and antisense sequences may be completelycomplementary across a substantial portion of their length. In otherembodiments the sense sequence and antisense sequence may be at least70, 80, 90, 95 or 99% complementarity across independently at least 50,60, 70, 80, 85, 90, 95, or 99% of the length of the strands.

Neither the identity of the sense sequence nor the homology of theantisense sequence need to be 100% complementarity to the targetsequence.

In one embodiment, separating the sense and antisense sequence of thestem loop structure of the modulatory polynucleotide is a loop sequence(also known as a loop motif, linker or linker motif). The loop sequencemay be of any length, between 4-30 nucleotides, between 4-20nucleotides, between 4-15 nucleotides, between 5-15 nucleotides, between6-12 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13nucleotides, 14 nucleotides, and/or 15 nucleotides.

In some embodiments, the loop sequence comprises a nucleic acid sequenceencoding at least one UGUG motif. In some embodiments, the nucleic acidsequence encoding the UGUG motif is located at the 5′ terminus of theloop sequence.

In one embodiment, spacer regions may be present in the modulatorypolynucleotide to separate one or more modules (e.g., 5′ flankingregion, loop motif region, 3′ flanking region, sense sequence, antisensesequence) from one another. There may be one or more such spacer regionspresent.

In one embodiment, a spacer region of between 8-20, i.e., 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides may be present betweenthe sense sequence and a flanking region sequence.

In one embodiment, the length of the spacer region is 13 nucleotides andis located between the 5′ terminus of the sense sequence and the 3′terminus of the flanking sequence. In one embodiment, a spacer is ofsufficient length to form approximately one helical turn of thesequence.

In one embodiment, a spacer region of between 8-20, i.e., 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides may be present betweenthe antisense sequence and a flanking sequence.

In one embodiment, the spacer sequence is between 10-13, i.e., 10, 11,12 or 13 nucleotides and is located between the 3′ terminus of theantisense sequence and the 5′ terminus of a flanking sequence. In oneembodiment, a spacer is of sufficient length to form approximately onehelical turn of the sequence.

In one embodiment, the molecular scaffold of the modulatorypolynucleotide comprises in the 5′ to 3′ direction, a 5′ flankingsequence, a 5′ arm., a loop motif, a 3′ arm and a 3′ flanking sequence.As a non-limiting example, the 5′ ann may comprise a nucleic acidsequence encoding a sense sequence and the 3′ arm comprises a nucleicacid sequence encoding the antisense sequence, In another non-limitingexample, the 5′ arm comprises a nucleic acid sequence encoding theantisense sequence and the 3′ arm comprises a nucleic acid sequenceencoding the sense sequence.

In one embodiment, the 5′ arm, sense and/or antisense sequence, loopmotif and/or 3′ aim sequence may be altered (e.g,, substituting 1 ormore nucleotides, adding nucleotides and/or deleting nucleotides). Thealteration may cause a beneficial change in the fimetion of theconstruct (e.g., increase knock-down of the target sequence, reducedegradation of the construct, reduce off target effect, increaseefficiency of the payload, and reduce degradation of the payload).

In one embodiment, the molecular scaffold of the modulatorypolynucleotides is aligned in order to have the rate of excision of theguide strand (also referred to herein as the antisense strand) begreater than the rate of excision of the passenger strand (also referredto herein as the sense strand). The rate of excision of the guide orpassenger strand may be, independently, 1%, 2%, 3%, 4%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 99% or more than 99%. As a non-limiting example, the rate ofexcision of the guide strand is at least 80%. As another non-limitingexample, the rate of excision of the guide strand is at least 90%.

In one embodiment, the rate of excision of the guide strand is greaterthan the rate of excision of the passenger strand. In one aspect, therate of excision of the guide strand may be at least 1%, 2%, 3%, 4%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 99% or more than 99% greater than the passengerstrand.

In one embodiment, the efficiency of excision of the guide strand is atleast 60%, 65%, 70%. 75%, 80%, 85%. 90%, 95%, 99% or more than 99%. As anon-limiting example, the efficiency of the excision of the guide strandis greater than 80%.

In one embodiment, the efficiency of the excision of the guide strand isgreater than the excision of the passenger strand from the molecularscaffold. The excision of the guide strand may be 2, 3, 4, 5, 6, 7, 8,9, 10 or more than 10 times more efficient than the excision of thepassenger strand from the molecular scaffold.

In one embodiment, the molecular scaffold comprises a dual-functiontargeting modulatory polynucleotide. As used herein, a “dual-fimetiontargeting” modulatory polynucleotide is a polynucleotide where both theguide and passenger strands knock down the same target or the guide andpassenger strands knock down different targets.

In one embodiment, the molecular scaffold of the modulatorypolynucleotides described herein may comprise a 5′ flanking region, aloop motif region and a 3′ flanking reaion. Non-limiting examples of thesequences for the 5′ flanking region, loop motif region (may also bereferred to as a linker region) and the 3′ flanking region which may beused, or fragments thereof used, in the modulatory polynucleotidesdescribed herein are shown in Tables 5-7.

TABLE 5  5′ Flanking Regions for Molecular Scaffold 5′ Flanking 5′Flanking Region Region Name 5′ Flanking Region Sequence SEQ ID 5F1CTCCCGCAGAACACCATGCGCTCCACG 3 GAA

TABLE 6  Loop Motif Regions for Molecular Scaffold Loop Motif Loop MotifLoop motif Region Name Region Sequence Region SEQ ID L1 GTGGCCACTGAGAAG4

TABLE 7  3′ Flanking Regions for Molecular Scaffold 3′ Flanking 3′Flanking 3′ Flanking Region Name Region Sequence Region SEQ ID 3F1CTGAGGAGCGCCTTGACA 5 GCAGCCATGGGAGGGCC

In one embodiment, the molecular scaffold may comprise at least one 5′flanking region, fragment or variant thereof listed in Table 5. As anon-limiting example, the 5′ flanking region may be 5F1.

In one embodiment, the molecular scaffold may comprise at least one 5F1flanking region.

In one embodiment, the molecular scaffold may comprise at least one loopmotif region, fragment or variant thereof listed in Table 6. As anon-limiting example, the loop motif region may be L1.

In one embodiment, the molecularscaffold may comprise at east one L1loop motif region.

In one embodiment, the molecular scaffold may comprise at least one 3′flanking region, fragment or variant thereof listed in Table 7. As anon-limiting example, the 3′ flanking region may be 3F1.

In one embodiment, the molecular scaffold may comprise at least one 3F1flanking region.

In one embodiment, the molecular scaffold may comprise at least one 5′flanking region, fragment or variant thereof, and at least one loopmotif region, fragment or variant thereof, as described in Tables 5 and6. As a non-limiting example, the 5′ flanking region and the loop motifregion may be 5F1 and L1.

In one embodiment, the molecular scaffold may comprise at least one 3′flanking region, fragment or variant thereof, and at least one motifregion, fragment or variant thereof, as described in Tables 6 and 7. Asa non-limiting example, the 3′ flanking region and the loop motif regionmay be 3F1 and L1.

In one embodiment, the molecular scaffold may comprise at least one 5′flanking region, fragment or variant thereof, and at least one 3′flanking region, fragment or variant thereof, as described in Tables 5and 7. As a non-limiting example, the flanking regions may be SF1 and3F1.

In one embodiment, the molecular scaffold may comprise at least one 5′flanking region, fragment or variant thereof, at least one loop motifregion, fragment or variant thereof, and at least one 3′ flanking regionas described in Tables 5-7. As a non-limiting example, the flanking andloop motif regions may be SF1, L1 and 3F1.

In one embodiment, the molecular scaffold may be a natural pri-miRNAscaffold. As a non-limiting example, the molecular scaffold may be ascaffold derived from the human miR155 scaffold.

In one embodiment, the molecular scaffold may comprise one or morelinkers known in the art. The linkers may separate regions or onemolecular scaffold from another. As a non-limiting example, themolecular scaffold may be polycistronic.

Modulatory Polynucleotede Comprising Molecular Scaffold and siRNAMolecules Targeting SOD1

In one embodiment, the modulatory polynucleotide may comprise 5′ and 3′flanking regions, loop motif region, and nucleic acid sequences encodingsense sequence and antisense sequence as described in Table 8. In Table8, the DNA sequence identifier for the passenger and guide strands aredescribed as well as the 5′ and 3′ Flanking Regions and the Loop region(also referred to as the linker region). In Table 8, the “miR” componentof the name of the sequence does not necessarily correspond to thesequence numbering of miRNA genes (e.g., VOY SOD1miR-102 is the name ofthe sequence and does not necessarily mean that miR-102 is part of thesequence).

TABLE 8 SOD1 Modulatory Polynucleotide Sequence Regions (5′ to 3′) 5′Flanking Modulatory to Polynucleotide 3′ Flanking 5′ Flanking PassengerLoop Guide 3′ Flanking Construct Name SEQ ID NO SEQ ID NO SEQ ID NO SEQID NO SEQ ID NO SEQ ID NO VOYSOD1miR104- 6 3 7 4 8 5 788.2

AAV Particles Comprising Modulatory Polynucleotides

In one embodiment, the AAV particle comprises a viral genome with apayload region comprising a modulatory polynucleotide sequence. In suchan embodiment, a viral genome encoding more than one polypeptide may bereplicated and packaged into a viral particle. A target cell transducedwith a viral particle comprising a modulatory polynucleotide may expressthe encoded sense and/or antisense sequences in a single cell.

In some embodiments, the AAV particles are useful in the field ofmedicine for the treatment, prophylaxis, palliation or amelioration ofneurological diseases and/or disorders,

In one embodiment, the AAV particles comprising modulatorypolynucleotide sequence which comprises a nucleic acid sequence encodingat least one siRNA molecule may be introduced into mammalian cells.

Where the AAV particle payload region comprises a modulatorypolynucleotide, the modulatory polynucleotide may comprise sense and/orantisense sequences to knock down a target gene. The AAV viral genomesencoding modulatory polynucleotides described herein may be useful inthe fields of human disease, viruses, infections veterinary applicationsand a variety of in vivo and in vitro settings.

In one embodiment, the AAV particle viral genome may comprise at leastone inverted terminal repeat (ITR) region. The ITR region(s) may,independently, have a length such as, but not limited to, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135. 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, and 175 nucleotides. The lengthof the ITR region for the viral genome may be 75-80, 75-85, 75-100,80-85, 80-90, 80-105, 85-90, 85-95, 85-110, 90-95, 90-100, 90-115,95-100, 95-105, 95-120, 100-105, 100-110, 100-125, 105-110, 105-115,105-130, 110-115, 110-120, 110-135, 115-120, 115-125, 115-140, 120-125,120-130, 120-145, 125-130, 125-135, 125-150, 130-135, 130-140, 130-155,135-140, 135-145, 135-160, 140-145, 140-150, 140-165, 145-150, 145-155,145-170, 150-155, 150-160, 150-175, 155-160, 155-165, 160-165, 160-170,165-170, 165-175, and 170-175 nucleotides. As a non-limiting example,the viral genome comprises an ITR that is about 105 nucleotides inlength. As a non-limiting, example, the viral genom.e comprises an ITRthat is about 141 nucleotides in length. As a non-limiting example, theviral genome comprises an ITR that is about 130 nucleotides in length.

In one embodiment, the AAV particle viral genome may comprises twoinverted terminal repeat (ITR) regions. Each of the ITR, regions mayindependently have a length such as, but not limited to, 75, 76, 77, 78,79. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93. 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 12.5,126, 127, 128, 129, 130, 131, 132, 133, 134. 135, 136, 137, 138, 139,140, 141, 142, 143. 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, and 175 nucleotides. The length ofthe ITR reaions for the viral genome may be 75-80, 75-85, 75-100, 80-85,80-90, 80-105, 85-90, 85-95, 85-110, 90-5 95, 90-100, 90-115, 95-100,95-105, 95-120, 100-105, 100-110, 100-125, 105-110, 105-115, 105-130,110-115, 110-120, 110-135, 115-120, 115-125, 115-140, 120-125, 120-130,120-145, 125-130, 125-135, 125-150, 130-135, 130-140, 130-155, 135-140,135-145, 135-160, 140-145, 140-150, 140-165, 145-150, 145-155, 145-170,150-155, 150-160, 150-175, 155-160, 155-165, 160-165, 160-170, 165-170,165-175, arid 170-175 nucleotides. As a non-limiting example, the viralgenome comprises an ITR that is about 105 nucleotides in length and 141nucleotides in length. As a non-limiting example, the viral genomecomprises an ITR that is about 105 nucleotides in length and 130nucleotides in length. As a non-limiting example, the viral genomecomprises an ITR that is about 130 nucleotides in length and 141nucleotides in length.

In one embodiment, the AAV particle viral genome comprises two ITRsequence regions.

In one embodiment, the AAV particle viral genome may comprise at leastone multiple filler sequence region. The filler region(s) may,independently, have a length such as, but not limited to, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412,413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426,427, 428, 429, 430, 431, 432, 4,33, 434, 435, 436, 437, 438, 439, 440,441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454,455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468,469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482,483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496,497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510,511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524,525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538,539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552,553, 554, 555, 556, 557 558, 559, 560, 561, 562, 563, 564, 565, 566,567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580,581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594,595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608,609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622,623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636,637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650,651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664,665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678,679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692,693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706,707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720,721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734,735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748,749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762,763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776,777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790,791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804,805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818,819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832,833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846,847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860,861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874,875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888,889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902,903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916,917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930,931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944,945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958,959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972,973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986,987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000,1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012,1013, 1014, 1015, 1016, 1017, 1018, 1019,1020, 1021, 1022, 1023, 1024,1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036,1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048,1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060,1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072,1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084,1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096,1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108,1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120,1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132,1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144,1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156,1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168,1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176, 1,177, 1178, 1179, 1180,1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192,1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204,1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216,1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228,1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240,1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252,1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264,1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276,1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288,1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300,1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312,1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324,1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336,1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348,1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360,1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372,1373, 1374, 1375, 1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384,1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396,1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404, 1405, 1406, 1407, 1408,1409, 1410, 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419, 1420,1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432,1433, 1434, 1435, 1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444,1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456,1457, 1458, 1459, 1460, 1461, 1462, 1463, 1464, 1465, 1466, 1467, 1468,1469, 1470, 1471, 1472, 1473, 1474, 1475, 1476, 1477, 1478, 1479, 1480,1481, 1482, 1483, 1484, 1485, 1486, 1487, 1488, 1489, 1490, 1491, 1492,1493, 1494, 1495, 1496, 1497, 1498, 1499, 1500, 1501, 1502, 1503, 1504,1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516,1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525, 1526, 1527, 1528,1529, 1530, 1531, 1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540,1541, 1542, 1543, 1544, 1545, 1546, 1547, 1548, 1549, 1550, 1551, 1552,1553, 1554, 1555, 1556, 1557, 1558, 1559, 1560, 1561, 1562, 1563, 1564,1565, 1566, 1567, 1568, 1569, 1570, 1571, 1572, 1573, 1574, 1575, 1576,1577, 1578, 1579, 1580, 1581, 1582, 1583, 1584, 1585, 1586, 1587, 1588,1589, 1590, 1591, 1592, 1593, 1594, 1595, 1596, 1597, 1598, 1599, 1600,1601, 1602, 1603, 1604, 1605, 1606, 1607, 1608, 1609, 1610, 1611, 1612,1613, 1614, 1615, 1616, 1617, 1618, 1619, 1620, 1621, 1622, 1623, 1624,1625, 1626, 1627, 1628, 1629, 1630, 1631, 1632, 1633, 1634, 1635, 1636,1637, 1638, 1639, 1640, 1641, 1642, 1643, 1644, 1645, 1646, 1647, 1648,1649, 1650, 1651, 1652, 1653, 1654, 1655, 1656, 1657, 1658, 1659, 1660,1661, 1662, 1663, 1664, 1665, 1666, 1667, 1668, 1669, 1670, 1671, 1672,1673, 1674, 1675, 1676, 1677, 1678, 1679, 1680, 1681, 1682, 1683, 1684,1685, 1686, 1687, 1688, 1689, 1690, 1691, 1692, 1693, 1694, 1695, 1696,1697, 1698, 1699, 1700, 1701, 1702, 1703, 1704, 1705, 1706, 1707, 1708,1709, 1710, 1711, 1712, 1713, 1714, 1715, 1716, 1717, 1718, 1719, 1720,1721, 1722, 1723, 1724, 1725, 1726, 1727, 1728, 1729, 1730, 1731, 1732,1733, 1734, 1735, 1736, 1737, 1738, 1739, 1740, 1741, 1742, 1743, 1744,1745, 1746, 1747, 1748, 1749, 1750, 1751, 1752, 1753, 1754, 1755, 1756,1757, 1758, 1759, 1760, 1761, 1762, 1763, 1764, 1765, 1766, 1767, 1768,1769, 1770, 1771, 1772, 1773, 1774, 1775, 1776, 1777, 1778, 1779, 1780,1781, 1782, 1783, 1784, 1785, 1786, 1787, 1788, 1789, 1790, 1791, 1792,1793, 1794, 1795, 1796, 1797, 1798, 1799, 1800, 1801, 1802, 1803, 1804,1805, 1806, 1807, 1808, 1809, 1810, 1811, 1812, 1813, 1814, 1815, 1816,1817, 1818, 1819, 1820, 1821, 1822, 1823, 1824, 1825, 1826, 1827, 1828,1829, 1830, 1831, 1832, 1833, 1834, 1835, 1836, 1837, 1838, 1839, 1840,1841, 1842, 1843, 1844, 1845, 1846, 1847, 1848, 1849, 1850, 1851, 1852,1853, 1854, 1855, 1856, 1857, 1858, 1859, 1860, 1861, 1862, 1863, 1864,1865, 1866, 1867, 1868, 1869, 1870, 1871, 1872, 1873, 1874, 1875, 1876,1877, 1878, 1879, 1880, 1881, 1882, 1883, 1884, 1885, 1886, 1887, 1888,1889, 1890, 1891, 1892, 1893, 1894, 1895, 1896, 1897, 1898, 1899, 1900,1901, 1902, 1903, 1904, 1905, 1906, 1907, 1908, 1909, 1910, 1911, 1912,1913, 1914, 1915, 1916, 1917, 1918, 1919, 1920, 1921, 1922, 1923, 1924,1925, 1926, 1927, 1928, 1929, 1930, 1931, 1932, 1933, 1934, 1935, 1936,1937, 1938, 1939, 1940, 1941, 1942, 1943, 1944, 1945, 1946, 1947, 1948,1949, 1950, 1951, 1952, 1953, 1954, 1955, 1956, 1957, 1958, 1959, 1960,1961, 1962, 1963, 1964, 1965, 1966, 1967, 1968, 1969, 1970, 1971, 1972,1973, 1974, 1975, 1976, 1977, 1978, 1979, 1980, 1981, 1982, 1983, 1984,1985, 1986, 1987, 1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995, 1996,1997, 1998, 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008,2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020,2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032,2033, 2034, 2035, 2036, 2037, 2038, 2039, 2040, 2041, 2042, 2043, 2044,2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056,2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068,2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080,2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092,2093, 2094, 2095, 2096, 2097, 2098, 2099, 2100, 2101, 2102, 2103, 2104,2105, 2106, 2107, 2108, 2109, 2110,2111, 2112, 2113, 2114, 2115, 2116,2117, 2118, 2119, 2120, 2121, 2122, 2123, 2124, 2125, 2126, 2127, 2128,2129, 2130, 2131, 2132, 2133, 2134, 2135, 2136, 2137, 2138, 2139, 2140,2141, 2142, 2143, 2144, 2145, 2146, 2147, 2148, 2149, 2150, 2151, 2152,2153, 2154, 2155, 2156, 2157, 2158, 2159, 2160, 2161, 2162, 2163, 2164,2165, 2166, 2167, 2168, 2169, 2170, 2171, 2172, 2173, 2174, 2175, 2176,2177, 2178, 2179, 2180, 2181, 2182, 2183, 2184, 2185, 2186, 2187, 2188,2189, 2190, 2191, 2192, 2193, 2194, 2195, 2196, 2197, 2198, 2199, 2200,2201, 2202, 2203, 2204, 2205, 2206, 2207, 2208, 2209, 2210, 2211, 2212,2213, 2214, 2215, 2216, 2217, 2218, 2219, 2220, 2221, 2222, 2223, 2224,2225, 2226, 2227, 2228, 2229, 2230, 2231, 2232, 2233, 2231, 2235, 2236,2237, 2238, 2239, 2240, 2241, 2242, 2243, 2244, 2245, 2246, 2247, 2248,2249, 2250, 2251, 2252, 2253, 2254, 2255, 2256, 2257, 2258, 2259, 2260,2261, 2262, 2263, 2264, 2265, 2266, 2267, 2268, 2269, 2270, 2271, 2272,2273, 2274, 2275, 2276, 2277, 2278, 2279, 2280, 2281, 2282, 2283, 2284,2285, 2286, 2287, 2288, 2289, 2290, 2291, 2292, 2293, 2294, 2295, 2296,2297, 2298, 2299, 2300, 2301, 2302, 2303, 2304, 2305, 2306, 2307, 2308,2309, 2310, 2311, 2312, 2313, 2314, 2315, 2316, 2317, 2318, 2319, 2320,2321, 2322, 2323, 2324, 2325, 2326, 2327, 2328, 2329, 2330, 2331, 2332,2333, 2334, 2335, 2336, 2337, 2338, 2339, 2340, 2341, 2342, 2343, 2344,2345, 2346, 2347, 2348, 2349, 2350, 2351, 2352, 2353, 2354, 2355, 2356,2357, 2358, 2359, 2360, 2361, 2362, 2363, 2364, 2365, 2366, 2367, 2368,2369, 2370, 2371, 2372, 2373, 2374, 2375, 2376, 2377, 2378, 2379, 2380,2381, 2382, 2383, 2384, 2385, 2386, 2387, 2388, 2389, 2390, 2391, 2392,2393, 2394, 2395, 2396, 2397, 2398, 2399, 2400, 2401, 2402, 2403, 2404,2405, 2406, 2407, 2408, 2409, 2410, 2411, 2412, 2413, 2414, 2415, 2416,2417, 2418, 2419, 2420, 2421, 2422, 2423, 2424, 2425, 2426, 2427, 2428,2429, 2430, 2431, 2432, 2433, 2434, 2435, 2436, 2437, 2438, 2439, 2440,2441, 2442, 2443, 2444, 2445, 2446, 2447, 2448, 2449, 2450, 2451, 2452,2453, 2454, 2455, 2456, 2457, 2458, 2459, 2460, 2461, 2462, 2463, 2464,2465, 2466, 2467, 2468, 2469, 2470, 2471, 2472, 2473, 2474, 2475, 2476,2477, 2478, 2479, 2480, 2481, 2482, 2483, 2484, 2485, 2486, 2487, 2488,2489, 2490, 2491, 2492, 2493, 2494, 2495, 2496, 2497, 2498, 2499, 2500,2501, 2502, 2503, 2504, 2505, 2506, 2507, 2508, 2509, 2510, 2511, 2512,2513, 2514, 2515, 2516, 2517, 2518, 2519, 2520, 2521, 2522, 2523, 2524,2525, 2526, 2527, 2528, 2529, 2530, 2531, 2532, 2533, 2534, 2535, 2536,2537, 2538, 2539, 2540, 2541, 2542, 2543, 2544, 2545, 2546, 2547, 2548,2549, 2550, 2551, 2552, 2553, 2554, 2555, 2556, 2557, 2558, 2559, 2560,2561, 2562, 2563, 2564, 2565, 2566, 2567, 2568, 2569, 2570, 2571, 2572,2573, 2574, 2575, 2576, 2577, 2578, 2579, 2580, 2581, 2582, 2583, 2584,2585, 2586, 2587, 2588, 2589, 2590, 2591, 2592, 2593, 2594, 2595, 2596,2597, 2598, 2599, 2600, 2601, 2602, 2603, 2604, 2605, 2606, 2607, 2608,2609, 2610, 2611, 2612, 2613, 2614, 2615, 2616, 2617, 2618, 2619, 2620,2621, 2622, 2623, 2624, 2625, 2626, 2627, 2628, 2629, 2630, 2631, 2632,2633, 2634, 2635, 2636, 2637, 2638, 2639, 2640, 2641, 2642, 2643, 2644,2645, 2646, 2647, 2648, 2649, 2650, 2651, 2652, 2653, 2654, 2655, 2656,2657, 2658, 2659, 2660, 2661, 2662, 2663, 2664, 2665, 2666, 2667, 2668,2669, 2670, 2671, 2672, 2673, 2674, 2675, 2676, 2677, 2678, 2679, 2680,2681, 2682, 2683, 2684, 2685, 2686, 2687, 2688, 2689, 2690, 2691, 2692,2693, 2694, 2695, 2696, 2697, 2698, 2699, 2700, 2701, 2702, 2703, 2704,2705, 2706, 2707, 2708, 2709, 2710, 2711, 2712, 2713, 2714, 2715, 2716,2717, 2718, 2719, 2720, 2721, 2722, 2723, 2724, 2725, 2726, 2727, 2728,2729, 2730, 2731, 2732, 2733, 2734, 2735, 2736, 2737, 2738, 2739, 2740,2741, 2742, 2743, 2744, 2745, 2746, 2747, 2748, 2749, 2750, 2751, 2752,2753, 2754, 2755, 2756, 2757, 2758, 2759, 2760, 2761, 2762, 2763, 2764,2765, 2766, 2767, 2768, 2769, 2770, 2771, 2772, 2773, 2774, 2775, 2776,2777, 2778, 2779, 2780, 2781, 2782, 2783, 2784, 2785, 2786, 2787, 2788,2789, 2790, 2791, 2792, 2793, 2794, 2795, 2796, 2797, 2798, 2799, 2800,2801, 2802, 2803, 2804, 2805, 2806, 2807, 2808, 2809, 2810, 2811, 2812,2813, 2814, 2815, 2816, 2817, 2818, 2819, 2820, 2821, 2822, 2823, 2824,2825, 2826, 2827, 2828, 2829, 2830, 2831, 2832, 2833, 2834, 2835, 2836,2837, 2838, 2839, 2840, 2841, 2842, 2843, 2844, 2845, 2846, 2847, 2848,2849, 2850, 2851, 2852, 2853, 2854, 2855, 2856, 2857, 2858, 2859, 2860,2861, 2862, 2863, 2864, 2865, 2866, 2867, 2868, 2869, 2870, 2871, 2872,2873, 2874, 2875, 2876, 2877, 2878, 2879, 2880, 2881, 2882, 2883, 2884,2885, 2886, 2887, 2888, 2889, 2890, 2891, 2892, 2893, 2894, 2895, 2896,2897, 2898, 2899, 2900, 2901, 2902, 2903, 2904, 2905, 2906, 2907, 2908,2909, 2910, 2911, 2912, 2913, 2914, 2915, 2916, 2917, 2918, 2919, 2920,2921, 2922, 2923, 2924, 2925, 2926, 2927, 2928, 2929, 2930, 2931, 2932,2933, 2934, 2935, 2936, 2937, 2938, 2939, 2940, 2941, 2942, 2943, 2944,2945, 2946, 2947, 2948, 2949, 2950, 2951, 2952, 2953, 2954, 2955, 2956,2957, 2958, 2959, 2960, 2961, 2962, 2963, 2964, 2965, 2966, 2967, 2968,2969, 2970, 2971, 2972, 2973, 2974, 2975, 2976, 2977, 2978, 2979, 2980,2981, 2982, 2983, 2984, 2985, 2986, 2987, 2988, 2989, 2990, 2991, 2992,2993, 2994, 2995, 2996, 2997, 2998, 2999, 3000, 3001, 3002, 3003, 3004,3005, 3006, 3007, 3008, 3009, 3010, 3011, 3012, 3013, 3014, 3015, 3016,3017, 3018, 3019, 3020, 3021, 3022, 3023, 3024, 3025, 3026, 3027, 3028,3029, 3030, 3031, 3032, 3033, 3034, 3035, 3036, 3037, 3038, 3039, 3040,3041, 3042, 3043, 3044, 3045, 3046, 3047, 3048, 3049, 3050, 3051, 3052,3053, 3054, 3055, 3056, 3057, 3058, 3059, 3060, 3061, 3062, 3063, 3064,3065, 3066, 3067, 3068, 3069, 3070, 3071, 3072, 3073, 3074, 3075, 3076,3077, 3078, 3079, 3080, 3081, 3082, 3083, 3084, 3085, 3086, 3087, 3088,3089, 3090, 3091, 3092, 3093, 3094, 3095, 3096, 3097, 3098, 3099, 3100,3101, 3102, 3103, 3104, 3105, 3106, 3107, 3108, 3109, 3110, 3111, 3112,3113, 3114, 3115, 3116, 3117, 3118, 3119, 3120, 3121, 3122, 3123, 3124,3125, 3126, 3127, 3128, 3129, 3130, 3131, 3132, 3133, 3134, 3135, 3136,3137, 3138, 3139, 3140, 3141, 3142, 3143, 3144, 3145, 3146, 3147, 3148,3149, 3150, 3151, 3152, 3153, 3154, 3155, 3156, 3157, 3158, 3159, 3160,3161, 3162, 3163, 3164, 3165, 3166, 3167, 3168, 3169, 3170, 3171, 3172,3173, 3174, 3175, 3176, 3177, 3178, 3179, 3180, 3181, 3182, 3183, 3184,3185, 3186, 3187, 3188, 3189, 3190, 3191, 3192, 3193, 3194, 3195, 3196,3197, 3198, 3199, 3200, 3201, 3202, 3203, 3204, 3205, 3206, 3207, 3208,3209, 3210, 3211, 3212, 3213, 3214, 3215, 3216, 3217, 3218, 3219, 3220,3221, 3222, 3223, 3224, 3225, 3226, 3227, 3228, 3229, 3230, 3231, 3232,3233, 3234, 3235, 3236, 3237, 3238, 3239, 3240, 3241, 3242, 3243, 3244,3245, 3246, 3247, 3248, 3249, and 3250 nucleotides, The length of anyfiller region for the viral genome may be 50-100, 100-150, 150-200,200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600,600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000,1000-1050, 1050-1100, 1100-1150, 1150-1200, 1200-1250, 1250-1300,1300-1350, 1350-1400, 1400-1450, 1450-1500, 1500-1550, 1550-1600,1600-1650, 1650-1700, 1700-1750, 1750-1800, 1800-1850, 1850-1900,1900-1950, 1950-2000, 2000-2050, 2050-2100, 2100-2150, 2150-2200,2200-2250, 2250-2300, 2300-2350, 2350-2400, 2400-2450, 2450-2500,2500-2550, 2550-2600, 2600-2650, 2650-2700, 2700-2750, 2750-2800,2800-2850, 2850-2900, 2900-2950, 2950-3000, 3000-3050, 3050-3100,3100-3150, 3150-3200, and 3200-3250 nucleotides. As a non-limitingexample, the viral genome comprises a filler region that is about 55nucleotides in length. As a non-limiting example, the viral genomecomprises a filler region that is about 56 nucleotides in length. As anon-limiting example, the viral genome comprises a filler region that isabout 97 nucleotides in length. As a non-limiting example, the viralgenome comprises a filler region that is about 103 nucleotides inlength. As a non-limiting example, the viral genome comprises a fillerregion that is about 105 nucleotides in length. As a non-limitingexample, the viral genome comprises a filler region that is about 357nucleotides in length. As a non-limiting example, the viral genomecomprises a filler region that is about 363 nucleotides in length. As anon-limiting example, the viral genome comprises a filler region that isabout 712 nucleotides in length. As a non-limiting example, the viralgenome comprises a filler region that is about 714 nucleotides inlength. As a non-limiting example, the viral genome comprises a fillerregion that is about 1203 nucleotides in length. As a non-limitingexample, the viral genome comprises a filler region that is about 1209nucleotides in length. As a non-limiting example, the viral genomecomprises a filler region that is about 1512 nucleotides in length. As anon-limiting example, the viral genome comprises a filler region that isabout 1519 nucleotides in length. As a non-limiting example, the viralgenome comprises a filler region that is about 2395 nucleotides inlength. As a non-limiting example, the viral genome comprises a fillerregion that is about 2403 nucleotides in length. As a non-limitingexample, the viral genome comprises a filler region that is about 2405nucleotides in length. As a non-limiting example, the viral genomecomprises a filler region that is about 3013 nucleotides in length. As anon-limiting example, the viral genome comprises a filler region that isabout 3021 nucleotides in length.

In one embodiment, the AAV particle viral genome may comprise at leastone multiple filler sequence region. The filler region(s) may,independently, have a length such as, but not limited to, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412,413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426,427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454,455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468,469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482,483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496,497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510,511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524,525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538,539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552,553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566,567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580,581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594,595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608,609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622,623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636,637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650,651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664,665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678,679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692,693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706,707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720,721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734,735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748,749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762,763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776,777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790,791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804,805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818,819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832,833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846,847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860,861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874,875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888,889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902,903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916,917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930,931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944,945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958,959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972,973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986,987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000,1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012,1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024,1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036,1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048,1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060,1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072,1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084,1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096,1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108,1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120,1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132,1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144,1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156,1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168,1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180,1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192,1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204,1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216,1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228,1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240,1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252,1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264,1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276,1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288,1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300,1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312,1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324,1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336,1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348,1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360,1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372,1373, 1374, 1375, 1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384,1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396,1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404, 1405, 1406, 1407, 1408,1409, 1410, 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419, 1420,1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432,1433, 1434, 1435, 1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444,1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456,1457, 1458, 1459, 1460, 1461, 1462, 1463, 1464, 1465, 1466, 1467, 1468,1469, 1470, 1471, 1472, 1473, 1474, 1475, 1476, 1477, 1478, 1479, 1480,1481, 1482, 1483, 1484, 1485, 1486, 1487, 1488, 1489, 1490, 1491, 1492,1493, 1494, 1495, 1496, 1497, 1498, 1499, 1500, 1501, 1502, 1503, 1504,1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516,1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525, 1526, 1527, 1528,1529, 1530, 1531, 1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540,1541, 1542, 1543, 1544, 1545, 1546, 1547, 1548, 1549, 1550, 1551, 1552,1553, 1554, 1555, 1556, 1557, 1558, 1559, 1560, 1561, 1562, 1563, 1564,1565, 1566, 1567, 1568, 1569, 1570, 1571, 1572, 1573, 1574, 1575, 1576,1577, 1578, 1579, 1580, 1581, 1582, 1583, 1584, 1585, 1586, 1587, 1588,1589, 1590, 1591, 1592, 1593, 1594, 1595, 1596, 1597, 1598, 1599, 1600,1601, 1602, 1603, 1604, 1605, 1606, 1607, 1608, 1609, 1610, 1611, 1612,1613, 1614, 1615, 1616, 1617, 1618, 1619, 1620, 1621, 1622, 1623, 1624,1625, 1626, 1627, 1628, 1629, 1630, 1631, 1632, 1633, 1634, 1635, 1636,1637, 1638, 1639, 1640, 1641, 1642, 1643, 1644, 1645, 1646, 1647, 1648,1649, 1650, 1651, 1652, 1653, 1654, 1655, 1656, 1657, 1658, 1659, 1660,1661, 1662, 1663, 1664, 1665, 1666, 1667, 1668, 1669, 1670, 1671, 1672,1673, 1674, 1675, 1676, 1677, 1678, 1679, 1680, 1681, 1682, 1683, 1684,1685, 1686, 1687, 1688, 1689, 1690, 1691, 1692, 1693, 1694, 1695, 1696,1697, 1698, 1699, 1700, 1701, 1702, 1703, 1704, 1705, 1706, 1707, 1708,1709, 1710, 1711, 1712, 1713, 1714, 1715, 1716, 1717, 1718, 1719, 1720,1721, 1722, 1723, 1724, 1725, 1726, 1727, 1728, 1729, 1730, 1731, 1732,1733, 1734, 1735, 1736, 1737, 1738, 1739, 1740, 1741, 1742, 1743, 1744,1745, 1746, 1747, 1748, 1749, 1750, 1751, 1752, 1753, 1754, 1755, 1756,1757, 1758, 1759, 1760, 1761, 1762, 1763, 1764, 1765, 1766, 1767, 1768,1769, 1770, 1771, 1772, 1773, 1774, 1775, 1776, 1777, 1778, 1779, 1780,1781, 1782, 1783, 1784, 1785, 1786, 1787, 1788, 1789, 1790, 1791, 1792,1793, 1794, 1795, 1796, 1797, 1798, 1799, 1800, 1801, 1802, 1803, 1804,1805, 1806, 1807, 1808, 1809, 1810, 1811, 1812, 1813, 1814, 1815, 1816,1817, 1818, 1819, 1820, 1821, 1822, 1823, 1824, 1825, 1826, 1827, 1828,1829, 1830, 1831, 1832, 1833, 1834, 1835, 1836, 1837, 1838, 1839, 1840,1841, 1842, 1843, 1844, 1845, 1846, 1847, 1848, 1849, 1850, 1851, 1852,1853, 1854, 1855, 1856, 1857, 1858, 1859, 1860, 1861, 1862, 1863, 1864,1865, 1866, 1867, 1868, 1869, 1870, 1871, 1872, 1873, 1874, 1875, 1876,1877, 1878, 1879, 1880, 1881, 1882, 1883, 1884, 1885, 1886, 1887, 1888,1889, 1890, 1891, 1892, 1893, 1894, 1895, 1896, 1897, 1898, 1899, 1900,1901, 1902, 1903, 1904, 1905, 1906, 1907, 1908, 1909, 1910, 1911, 1912,1913, 1914, 1915, 1916, 1917, 1918, 1919, 1920, 1921, 1922, 1923, 1924,1925, 1926, 1927, 1928, 1929, 1930, 1931, 1932, 1933, 1934, 1935, 1936,1937, 1938, 1939, 1940, 1941, 1942, 1943, 1944, 1945, 1946, 1947, 1948,1949, 1950, 1951, 1952, 1953, 1954, 1955, 1956, 1957, 1958, 1959, 1960,1961, 1962, 1963, 1964, 1965, 1966, 1967, 1968, 1969, 1970, 1971, 1972,1973, 1974, 1975, 1976, 1977, 1978, 1979, 1980, 1981, 1982, 1983, 1984,1985, 1986, 1987, 1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995, 1996,1997, 1998, 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008,2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020,2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032,2033, 2034, 2035, 2036, 2037, 2038, 2039, 2040, 2041, 2042, 2043, 2044,2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056,2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068,2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080,2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092,2093, 2094, 2095, 2096, 2097, 2098, 2099, 2100, 2101, 2102, 2103, 2104,2105, 2106, 2107, 2108, 2109, 2110, 2111, 2112, 2113, 2114, 2115, 2116,2117, 2118, 2119, 2120, 2121, 2122, 2123, 2124, 2125, 2126, 2127, 2128,2129, 2130, 2131, 2132, 2133, 2134, 2135, 2136, 2137, 2138, 2139, 2140,2141, 2142, 2143, 2144, 2145, 2146, 2147, 2148, 2149, 2150, 2151, 2152,2153, 2154, 2155, 2156, 2157, 2158, 2159, 2160, 2161, 2162, 2163, 2164,2165, 2166, 2167, 2168, 2169, 2170, 2171, 2172, 2173, 2174, 2175, 2176,2177, 2178, 2179, 2180, 2181, 2182, 2183, 2184, 2185, 2186, 2187, 2188,2189, 2190, 2191, 2192, 2193, 2194, 2195, 2196, 2197, 2198, 2199, 2200,2201, 2202, 2203, 2204, 2205, 2206, 2207, 2208, 2209, 2210, 2211, 2212,2213, 2214, 2215, 2216, 2217, 2218, 2219, 2220, 2221, 2222, 2223, 2224,2225, 2226, 2227, 2228, 2229, 2230, 2231, 2232, 2233, 2234, 2235, 2236,2237, 2238, 2239, 2240, 2241, 2242, 2243, 2244, 2245, 2246, 2247, 2248,2249, 2250, 2251, 2252, 2253, 2254, 2255, 2256, 2257, 2258, 2259, 2260,2261, 2262, 2263, 2264, 2265, 2266, 2267, 2268, 2269, 2270, 2271, 2272,2273, 2274, 2275, 2276, 2277, 2278, 2279, 2280, 2281, 2282, 2283, 2284,2285, 2286, 2287, 2288, 2289, 2290, 2291, 2292, 2293, 2294, 2295, 2296,2297, 2298, 2299, 2300, 2301, 2302, 2303, 2304, 2305, 2306, 2307, 2308,2309, 2310, 2311, 2312, 2313, 2314, 2315, 2316, 2317, 2318, 2319, 2320,2321, 2322, 2323, 2324, 2325, 2326, 2327, 2328, 2329, 2330, 2331, 2332,2333, 2334, 2335, 2336, 2337, 2338, 2339, 2340, 2341, 2342, 2343, 2344,2345, 2346, 2347, 2348, 2349, 2350, 2351, 2352, 2353, 2354, 2355, 2356,2357, 2358, 2359, 2360, 2361, 2362, 2363, 2364, 2365, 2366, 2367, 2368,2369, 2370, 2371, 2372, 2373, 2374, 2375, 2376, 2377, 2378, 2379, 2380,2381, 2382, 2383, 2384, 2385, 2386, 2387, 2388, 2389, 2390, 2391, 2392,2393, 2394, 2395, 2396, 2397, 2398, 2399, 2400, 2401, 2402, 2403, 2404,2405, 2406, 2407, 2408, 2409, 2410, 2411, 2412, 2413, 2414, 2415, 2416,2417, 2418, 2419, 2420, 2421, 2422, 2423, 2424, 2425, 2426, 2427, 2428,2429, 2430, 2431, 2432, 2433, 2434, 2435, 2436, 2437, 2438, 2439, 2440,2441, 2442, 2443, 2444, 2445, 2446, 2447, 2448, 2449, 2450, 2451, 2452,2453, 2454, 2455, 2456, 2457, 2458, 2459, 2460, 2461, 2462, 2463, 2464,2465, 2466, 2467, 2468, 2469, 2470, 2471, 2472, 2473, 2474, 2475, 2476,2477, 2478, 2479, 2480, 2481, 2482, 2483, 2484, 2485, 2486, 2487, 2488,2489, 2490, 2491, 2492, 2493, 2494, 2495, 2496, 2497, 2498, 2499, 2500,2501, 2502, 2503, 2504, 2505, 2506, 2507, 2508, 2509, 2510, 2511, 2512,2513, 2514, 2515, 2516, 2517, 2518, 2519, 2520, 2521, 2522, 2523, 2524,2525, 2526, 2527, 2528, 2529, 2530, 2531, 2532, 2533, 2534, 2535, 2536,2537, 2538, 2539, 2540, 2541, 2542, 2543, 2544, 2545, 2546, 2547, 2548,2549, 2550, 2551, 2552, 2553, 2554, 2555, 2556, 2557, 2558, 2559, 2560,2561, 2562, 2563, 2564, 2565, 2566, 2567, 2568, 2569, 2570, 2571, 2572,2573, 2574, 2575, 2576, 2577, 2578, 2579, 2580, 2581, 2582, 2583, 2584,2585, 2586, 2587, 2588, 2589, 2590, 2591, 2592, 2593, 2594, 2595, 2596,2597, 2598, 2599, 2600, 2601, 2602, 2603, 2604, 2605, 2606, 2607, 2608,2609, 2610, 2611, 2612, 2613, 2614, 2615, 2616, 2617, 2618, 2619, 2620,2621, 2622, 2623, 2624, 2625, 2626, 2627, 2628, 2629, 2630, 2631, 2632,2633, 2634, 2635, 2636, 2637, 2638, 2639, 2640, 2641, 2642, 2643, 2644,2645, 2646, 2647, 2648, 2649, 2650, 2651, 2652, 2653, 2654, 2655, 2656,2657, 2658, 2659, 2660, 2661, 2662, 2663, 2664, 2665, 2666, 2667, 2668,2669, 2670, 2671, 2672, 2673, 2674, 2675, 2676, 2677, 2678, 2679, 2680,2681, 2682, 2683, 2684, 2685, 2686, 2687, 2688, 2689, 2690, 2691, 2692,2693, 2694, 2695, 2696, 2697, 2698, 2699, 2700, 2701, 2702, 2703, 2704,2705, 2706, 2707, 2708, 2709, 2710, 2711, 2712, 2713, 2714, 2715, 2716,2717, 2718, 2719, 2720, 2721, 2722, 2723, 2724, 2725, 2726, 2727, 2728,2729, 2730, 2731, 2732, 2733, 2734, 2735, 2736, 2737, 2738, 2739, 2740,2741, 2742, 2743, 2744, 2745, 2746, 2747, 2748, 2749, 2750, 2751, 2752,2753, 2754, 2755, 2756, 2757, 2758, 2759, 2760, 2761, 2762, 2763, 2764,2765, 2766, 2767, 2768, 2769, 2770, 2771, 2772, 2773, 2774, 2775, 2776,2777, 2778, 2779, 2780, 2781, 2782, 2783, 2784, 2785, 2786, 2787, 2788,2789, 2790, 2791, 2792, 2793, 2794, 2795, 2796, 2797, 2798, 2799, 2800,2801, 2802, 2803, 2804, 2805, 2806, 2807, 2808, 2809, 2810, 2811, 2812,2813, 2814, 2815, 2816, 2817, 2818, 2819, 2820, 2821, 2822, 2823, 2824,2825, 2826, 2827, 2828, 2829, 2830, 2831, 2832, 2833, 2834, 2835, 2836,2837, 2838, 2839, 2840, 2841, 2842, 2843, 2844, 2845, 2846, 2847, 2848,2849, 2850, 2851, 2852, 2853, 2854, 2855, 2856, 2857, 2858, 2859, 2860,2861, 2862, 2863, 2864, 2865, 2866, 2867, 2868, 2869, 2870, 2871, 2872,2873, 2874, 2875, 2876, 2877, 2878, 2879, 2880, 2881, 2882, 2883, 2884,2885, 2886, 2887, 2888, 2889, 2890, 2891, 2892, 2893, 2894, 2895, 2896,2897, 2898, 2899, 2900, 2901, 2902, 2903, 2904, 2905, 2906, 2907, 2908,2909, 2910, 2911, 2912, 2913, 2914, 2915, 2916, 2917, 2918, 2919, 2920,2921, 2922, 2923, 2924, 2925, 2926, 2927, 2928, 2929, 2930, 2931, 2932,2933, 2934, 2935, 2936, 2937, 2938, 2939, 2940, 2941, 2942, 2943, 2944,2945, 2946, 2947, 2948, 2949, 2950, 2951, 2952, 2953, 2954, 2955, 2956,2957, 2958, 2959, 2960, 2961, 2962, 2963, 2964, 2965, 2966, 2967, 2968,2969, 2970, 2971, 2972, 2973, 2974, 2975, 2976, 2977, 2978, 2979, 2980,2981, 2982, 2983, 2984, 2985, 2986, 2987, 2988, 2989, 2990, 2991, 2992,2993, 2994, 2995, 2996, 2997, 2998, 2999, 3000, 3001, 3002, 3003, 3004,3005, 3006, 3007, 3008, 3009, 3010, 3011, 3012, 3013, 3014, 3015, 3016,3017, 3018, 3019, 3020, 3021, 3022, 3023, 3024, 3025, 3026, 3027, 3028,3029, 3030, 3031, 3032, 3033, 3034, 3035, 3036, 3037, 3038, 3039, 3040,3041, 3042, 3043, 3044, 3045, 3046, 3047, 3048, 3049, 3050, 3051, 3052,3053, 3054, 3055, 3056, 3057, 3058, 3059, 3060, 3061, 3062, 3063, 3064,3065, 3066, 3067, 3068, 3069, 3070, 3071, 3072, 3073, 3074, 3075, 3076,3077, 3078, 3079, 3080, 3081, 3082, 3083, 3084, 3085, 3086, 3087, 3088,3089, 3090, 3091, 3092, 3093, 3094, 3095, 3096, 3097, 3098, 3099, 3100,3101, 3102, 3103, 3104, 3105, 3106, 3107, 3108, 3109, 3110, 3111, 3112,3113, 3114, 3115, 3116, 3117, 3118, 3119, 3120, 3121, 3122, 3123, 3124,3125, 3126, 3127, 3128, 3129, 3130, 3131, 3132, 3133, 3134, 3135, 3136,3137, 3138, 3139, 3140, 3141, 3142, 3143, 3144, 3145, 3146, 3147, 3148,3149, 3150, 3151, 3152, 3153, 3154, 3155, 3156, 3157, 3158, 3159, 3160,3161, 3162, 3163, 3164, 3165, 3166, 3167, 3168, 3169, 3170, 3171, 3172,3173, 3174, 3175, 3176, 3177, 3178, 3179, 3180, 3181, 3182, 3183, 3184,3185, 3186, 3187, 3188, 3189, 3190, 3191, 3192, 3193, 3194, 3195, 3196,3197, 3198, 3199, 3200, 3201, 3202, 3203, 3204, 3205, 3206, 3207, 3208,3209, 3210, 3211, 3212, 3213, 3214, 3215, 3216, 3217, 3218, 3219, 3220,3221, 3222, 3223, 3224, 3225, 3226, 3227, 3228, 3229, 3230, 3231, 3232,3233, 3234, 3235, 3236, 3237, 3238, 3239, 3240, 3241, 3242, 3243, 3244,3245, 3246, 3247, 3248, 3249, and 3250 nucleotides. The length of anyfiller region for the viral genome may be 50-100, 100-150, 150-200,200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600,600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000,1000-1050, 1050-1100, 1100-1150, 1150-1200, 1200-1250, 1250-1300,1300-1350, 1350-1400, 1400-1450, 1450-1500, 1500-1550, 1550-1600,1600-1650, 1650-1700, 1700-1750, 1750-1800, 1800-1850, 1850-1900,1900-1950, 1950-2000, 2000-2050, 2050-2100, 2100-2150, 2150-2200,2200-2250, 2250-2300, 2300-2350, 2350-2400, 2400-2450, 2450-2500,2500-2550, 2550-2600, 2600-2650, 2650-2700, 2700-2750, 2750-2800,2800-2850, 2850-2900, 2900-2950, 2950-3000, 3000-3050, 3050-3100,3100-3150, 3150-3200, and 3200-3250 nucleotides. As a non-limitingexample, the viral izenome comprises a filler region that is about 55nucleotides in length. As a non-limiting example, the viral genomecomprises a filler region that is about 56 nucleotides in length. As anon-limiting example, the viral genome comprises a filler region that isabout 97 nucleotides in length. As a non-limiting example, the viralgenome comprises a filler region that is about 103 nucleotides inlength. As a non-limiting example, the viral genome comprises a fillerregion that is about 105 nucleotides in length. As a non-limitingexample, the viral genome comprises a filler region that is about 357nucleotides in length. As a non-limiting example, the viral genomecomprises a filler region that is about 363 nucleotides in length. As anon-limiting example, the viral genome comprises a filler region that isabout 712 nucleotides in length. As a non-limiting example, the viralgenome comprises a filler region that is about 714 nucleotides inlength. As a non-limiting example, the viral genome comprises a fillerregion that is about 1203 nucleotides in length. As a non-limitingexample, the viral genome comprises a filler region that is about 1209nucleotides in length. As a non-limiting example, the viral genomecomprises a filler region that is about 1512 nucleotides in length. As anon-limiting example, the viral genome comprises a filler region that isabout 1519 nucleotides in length. As a non-limiting example, the viralgenome comprises a filler region that is about 2395 nucleotides inlength. As a non-limiting example, the viral genome comprises a fillerregion that is about 2403 nucleotides in length. As a non-limitingexample, the viral genome comprises a filler region that is about 2405nucleotides in length. As a non-limiting example, the viral genomecomprises a filler region that is about 3013 nucleotides in length. As anon-limiting example, the viral genome comprises a filler region that isabout 3021 nucleotides in length.

In one embodiment, the AAV particle viral genome may comprise at leastone enhancer sequence region. The enhancer sequence region(s) may,independently, have a length such as, but not limited to, 300, 301, 302,303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316,317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330,331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344,345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358,359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372,373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386,387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, and 400nucleotides. The length of the enhancer region for the viral genome maybe 300-310, 300-325, 305-315, 310-320, 315-325, 320-330, 325-335,325-350, 330-340, 335-345, 340-350, 345-355, 350-360, 350-375, 355-365,360-370, 365-375, 370-380, 375-385, 375-400, 380-390, 385-395, and390-400 nucleotides. As a non-limiting example, the viral genomecomprises an enhancer region that is about 303 nucleotides in length. Asa non-limiting example, the viral genome comprises an enhancer regionthat is about 382 nucleotides in length.

In one embodiment, the AAV particle viral genome may comprise at leastone promoter sequence region. The promoter sequence region(s) may,independently, have a length such as, but not limited to, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52 53 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308,309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322,323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336,337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350,351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364,365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378,379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392,393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406,407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420,421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434,435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448,449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462,463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476,477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490,491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504,505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518,519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532,533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546,547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560,561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574,575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588,589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, and 600nucleotides. The length of the promoter region for the viral genome maybe 4-10, 10-20, 10-50, 20-30, 30-40, 40-50, 50-60, 50-100, 60-70, 70-80,80-90, 90-100, 100-110, 100-150, 110-120, 120-130, 130-140, 140-150,150-160, 150-200, 160-170, 170-180, 180-190, 190-200, 200-210, 200-250,210-220, 220-230, 230-240, 240-250, 250-260, 250-300, 260-270, 270-280,280-290, 290-300, 300-310, 300-350, 310-320, 320-330, 330-340, 340-350,350-360, 350-400, 360-370, 370-380, 380-390, 390-400, 400-410, 400-450,410-420, 420-430, 430-440, 440-450, 450-460, 450-500, 460-470, 470-480,480-490, 490-500, 500-510, 500-550, 510-520, 520-530, 530-540, 540-550,550-560, 550-600, 560-570, 570-580, 580-590, and 590-600 nucleotides. Asa non-limiting example, the viral genome comprises a promoter regionthat is about 4 nucleotides in length. As a non-limiting example, theviral genome comprises a promoter region that is about 17 nucleotides inlength. As a non-limiting example, the viral genome comprises a promoterregion that is about 204 nucleotides in length. As a non-limitingexample, the viral genome comprises a promoter region that is about 219nucleotides in length. As a non-limiting example, the viral genomecomprises a promoter region that is about 260 nucleotides in length. Asa non-limiting example, the viral genome comprises a promoter regionthat is about 303 nucleotides in length. As a non-limiting example, theviral genome comprises a promoter region that is about 382 nucleotidesin length. As a non-limiting example, the viral genome comprises apromoter region that is about 588 nucleotides in length.

In one embodiment, the AAV particle viral genome may comprise at leastone exon sequence region. The exon region(s) may, independently, have alength such as, but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, and 150 nucleotides. The length of theexon region for the viral genome may be 2-10, 5-10, 5-15, 10-20, 10-30,10-40, 15-20, 15-25, 20-30, 20-40, 20-50, 25-30, 25-35, 30-40, 30-50,30-60, 35-40, 35-45, 40-50, 40-60, 40-70, 45-50, 45-55, 50-60, 50-70,50-80, 55-60, 55-65, 60-70, 60-80, 60-90, 65-70, 65-75, 70-80, 70-90,70-100, 75-80, 75-85, 80-90, 80-100, 80-110, 85-90, 85-95, 90-100,90-110, 90-120, 95-100, 95-105, 100-110, 100-120, 100-130, 105-110,105-115, 110-120, 110-130, 110-140, 115-120, 115-125, 120-130, 120-140,120-150, 125-130, 125-135, 130-140, 130-150, 135-140, 135-145, 140-150,and 145-150 nucleotides. As a non-limiting example, the viral genomecomprises an exon region that is about 53 nucleotides in length. As anon-limiting example, the viral genome comprises an exon region that isabout 134 nucleotides in length.

In one embodiment, the AAV particle: genome may comprise at least oneintron sequence region. The intron region(s) may, independently, have alength such as, but not limited to, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 346, 347, 348, 349, and 350 nucleotides. The length ofthe intron region for the viral genome may be 25-35, 25-50, 35-45,45-55, 50-75, 55-65, 65-75, 75-85, 75-100, 85-95, 95-105, 100-125,105-115, 115-125, 125-135, 125-150, 135-145, 145-155, 150-175, 155-165,165-175, 175-185, 175-200, 185-195, 195-205, 200-225, 205-215, 215-225,225-235, 225-250, 235-245, 245-255, 250-275, 255-265, 265-275, 275-285,275-300, 285-295, 295-305, 300-325, 305-315, 315-325, 325-335, 325-350,and 335-345 nucleotides. As a non-limiting example, the viral genomecomprises an intron region that is about 32 nucleotides in length. As anon-limiting example, the viral genome comprises an intron region thatis about 172 nucleotides in length. As a non-limiting example, the viralgenome comprises an intron region that is about 201 nucleotides inlermth. As a non-limiting example, the viral genome comprises an intronregion that is about 347 nucleotides in length.

In one embodiment, the AAV particle viral genome may comprise at leastone polyadenylation signal sequence region. The polyadenylation signalregion sequence region(s) may, independently, have a length such as, butnot limited to, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176,177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190,191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204,205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246,247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260,261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274,275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288,289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302,303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316,317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330,331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344,345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358,359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372,373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386,387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400,401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414,415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428,429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442,443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456,457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470,471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484,485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498,499, 500 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512,513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526,527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540,541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554,555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568,569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582,583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596,597, 598, 599, and 600 nucleotides. The length of the polyadenylationsignal sequence region for the viral genome may be 4-10, 10-20, 10-50,20-30, 30-40, 40-50, 50-60, 50-100, 60-70, 70-80, 80-90, 90-100,100-110, 100-150, 110-120, 120-130, 130-140, 140-150, 150-160, 150-200,160-170, 170-180, 180-190, 190-200, 200-210, 200-250, 210-220, 220-230,230-240, 240-250, 250-260, 250-300, 260-270, 270-280, 280-290, 290-300,300-310, 300-350, 310-320, 320-330, 330-340, 340-350, 350-360, 350-400,360-370, 370-380, 380-390, 390-400, 400-410, 400-450, 410-420, 420-430,430-440, 440-450, 450-460, 450-500, 460-470, 470-480, 480-490, 490-500,500-510, 500-550, 510-520, 520-530, 530-540, 540-550, 550-560, 550-600,560-570, 570-580, 580-590, and 590-600 nucleotides. As a non-limitingexample, the viral genome comprises a polyadenylation signal sequenceregion that is about 127 nucleotides in length. As a non-limitingexample, the viral genome comprises a polyadenylation signal sequenceregion that is about 225 nucleotides in length. As a non-limitingexample, the viral genome comprises a polyadenylation signal sequenceregion that is about 476 nucleotides in length. As a non-limitingexample, the viral genome comprises a polyadenylation signal sequenceregion that is about 477 nucleotides in length.

In one embodiment, the AAV particle viral genome comprises more than onepolyA signal sequence region.

Non-limiting examples of ITR to ITR sequences of AAV particlescomprising a viral genome with a payload region comprising; a modulatorypolynucleotide sequence are described in Table 9. Table 9 also providesan alternate name for the ITR to ITR construct indicated by the “VOYSOD”identifier.

TABLE 9 ITR to ITR Sequences of AAV Particles comprising ModulatoryPolynucleotides Modulatory ITR to ITR ITR to ITR PolynucleotideConstruct Name SEQ ID NO SEQ ID NO VOYSOD16 9 6

In one embodiment, the AAV particle comprises a viral genome whichcomprises a sequence which has a percent identity to SEQ ID NO: 9. Theviral genome may have 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 99% or 100% identity to SEQ ID NO: 9. The viral genome may have1-10%, 10-20%, 30-40%, 50-60%, 50-70%, 50-80%, 50-90%, 50-99%, 50-100%,60-70%, 60-80%, 60-90%, 60-99%, 60-100%, 70-80%, 70-90%, 70-99%,70-100%, 80-85%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, or90-100% to SEQ ID NO: 9. As a non-limiting example, the viral genomecomprises a sequence which as 80% identity to SEQ ID NO: 9. As anothernon-limiting example, the viral genotne comprises a sequence which as85% identity to SEQ ID NO: 9. As another non-limiting example, the viralgenome comprises a sequence which as 90% identity to SEQ ID NO: 9. Asanother non-limiting example, the viral genotne comprises a sequencewhich as 95% identity to SEQ ID NO: 9. As another non-limiting example,the viral genome comprises a sequence which as 99% identity to SEQ NO:9.

AAV particles may be modified to enhance the efficiency of delivery.Such modified AAV particles comprising the nucleic acid sequenceencoding the siRNA molecules of the present disclosure can be packagedefficiently and can be used to successfully infect the target cells athigh frequency and with minimal toxicity.

In some embodiments, the AAV particle comprising a nucleic acid sequenceencoding the siRNA molecules of the present disclosure may be a humanserotype AAV particle. Such human AAV particle may be derived from anyknown serotype, e.g., from any one of serotypes AAV1-AAVIL Asnon-limiting examples. AAV particles may be vectors comprising anAAV1-derived genome in an AAV1-derived capsid; vectors comprising anAAV2-derived genome in an AAV2-derived capsid; vectors comprising anAAV4-derived genome in an AAV4 derived capsid; vectors comprising anAAV6-derived genome in an AAV6 derived capsid or vectors comprising anAAV9-derived genome in an AAV9 derived capsid.

In other embodiments, the AAV particle comprising a nucleic acidsequence for encoding siRNA molecules of the present disclosure may be apseudotyped hybrid or chimeric AAV particle which contains sequencesand/or components originating from at least two different AAV serotypes.Pseudotyped AAV particles may be vectors comprising an AAV genomederived from one AAV serotype and a capsid protein derived at least inpart from a different AAV serotype. As non-limiting examples, suchpseudotyped AAV particles may be vectors comprising an AAV2-derivedgenome in an AAV1-derived capsid; or vectors comprising an AAV2-derivedgenome in an AAV6-derived capsid; or vectors comprising an AAV2-derivedgenome in an AAV4-derived capsid; or an AAV2-derived genome in anAAV9-derived capsid. In like fashion, the present disclosurecontemplates any hybrid or chimeric AAV particle.

In other embodiments, AAV particles comprising a nucleic acid sequenceencoding the siRNA molecules of the present disclosure may be used todeliver siRNA molecules to the central nervous system (e.g., U.S. Pat.No. 6,180,613; the contents of which is herein incorporated by referencein its entirety).

In some aspects, the AAV particles comprising a nucleic acid sequenceencoding the siRNA molecules of the present disclosure may furthercomprise a modified capsid including peptides from non-viral origin. Inother aspects, the AAV particle may contain a CNS specific chimericcapsid to facilitate the delivery of encoded siRNA duplexes into thebrain and the spinal cord. For example, an alignment of cap nucleotidesequences from AAV variants exhibiting CNS tropism may be constructed toidentify variable region (VR) sequence and structure.

In other embodiments, the siRNA molecules of the present disclosure canbe encoded in plasmid vectors, viral vectors (e.g., AAV vectors), genomeor other nucleic acid expression vectors for delivery to a cell.

DNA expression plasmids can be used to stably express the siRNA duplexesor dsRNA of the present disclosure in cells and achieve long-terminhibition of target gene.

In one aspect, the sense and antisense strands of a siRNA duplex encodedby a SOD1 targeting polynucleotide are typically linked by a shortspacer sequence leading to the expression of a stein-loop stricturetermed short hairpin RNA (shRNA). The hairpin is recognized and cleavedby Dicer, thus generating mature siRNA molecules.

According to the present disclosure, AAV vectors comprising the nucleicacids of the siRNA. molecules targeting SOD1 mRNA are produced, the AAVvectors may be AAV AAV2, AAV3, AAV4, AANT5, AAV6, AAV7, AAV8, AAV9,AAV9,47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh AAV-DJ8 andAAV-DJ, and variants thereof.

In some embodiments, the siRNA duplexes or dsRNA of the presentdisclosure when expressed suppress (or degrade) target mRNA (i.e. SOD1).Accordingly, the siRNA duplexes or dsRNA encoded by a SOD1 targetingpolynucleotide can be used to substantially inhibit SOD1 gene expressionin a cell, for example a motor neuron. In some aspects, the inhibitionof SOD! gene expression refers to an inhibition by at least about 20%,preferably by at least about 30%, 40%, 50%, 60%, 70%, 80%. 85%, 90%, 95%and 100%. Accordingly, the protein product of the targeted gene may beinhibited by at least about 20%, preferably by at least about 30%, 40%,50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, The SOD1 gene can be eithera wild type gene or a mutated SOD1 gene with at least one mutation.Accordingly, the protein is either wild type protein or a mutatedpolypeptide with at least one mutation.

Viral Production

The present disclosure provides a method for the generation ofparvoviral particles, e.g. AAV particles, by viral genome replication ina viral replication cell comprising contacting the viral replicationcell with an AAV polynucleotide or AAV genome.

The present disclosure provides a method for producing an AAV particlehaving enhanced (increased, improved) transduction efficiency comprisingthe steps of: 1) co-transfecting competent bacterial cells with a bacmidvector and either a viral construct vector and/or AAV payload constructvector. 2) isolating the resultant viral construct expression vector andAAV payload construct expression vector and separately transfectingviral replication cells, 3) isolating and purifying resultant payloadand viral construct particles comprising viral construct expressionvector or AAV payload construct expression vector, 4) co-infecting aviral replication cell with both the AAV payload and viral constructparticles comprising viral construct expression vector or AAV payloadconstruct expression vector, 5) harvesting and purifying the viralparticle comprising a parvoviral genome.

In one embodiment, the present disclosure provides a method forproducing an AAV particle comprising the steps of 1) simultaneouslyco-transfecting mammalian cells, such as, but not limited to HEK293cells, with a payload region, a construct expressing rep and cap genesand a helper construct, 2) harvesting and purifying the AAV particlecomprising a viral genome.

Cells

The present disclosure provides a cell comprising an AAV polynucleotideand/or AAV genome.

Viral production disclosed herein describes processes and methods forproducing AAV particles that contact a target cell to deliver a payloadconstruct, e.g. a recombinant viral construct, which comprises apolynucleotide sequence encoding a payload molecule.

In one embodiment, the AAV particles may be produced in a viralreplication cell that comprises an insect cell.

Growing conditions for insect cells in culture, and production ofheterologous products in insect cells in culture are well-known in theart, see U.S. Pat. No. 6,204,059, the contents of which are hereinincorporated by reference in their entirety.

Any insect cell which allows for replication of parvovirus and which canbe maintained in culture can be used in accordance with the presentdisclosure. Cell lines may be used from Spodoptera frugiperda,including, but not limited to the Sf9 or Sf21 cell lines, Drosophilacell lines, or mosquito cell lines, such as Aedes albopictus derivedcell lines. Use of insect cells for expression of heterologous proteinsis well documented, as are methods of introducing nucleic acids, such asvectors, e.g., insect-cell compatible vectors, into such cells andmethods of maintaining such cells in culture. See, for example, Methodsin Molecular Biology, ed. Richard, Humana Press, NJ (1995); O'Reilly etal., Baculovirus Expression Vectors, A Laboratory Manual, Oxford Univ.Press (1994); Samulski et al., J. Vir. 63:3822-8 (1989); Kajigaya. etal., Proc. Nat'l. Acad. Sci. USA 88: 4646-50 (1991); Ruffing et al., J.Vir. 66:6922-30 (1992); Kimbauer et al., Vir. 219:37-44 (1996); Zhao etal., Vir.272:382-93 (2000); and Samulski et al., U.S. Pat. No.6,204,059, the contents of each of which is herein incorporated byreference in its entirety.

The viral replication cell may be selected from any biological organism,including prokaryotic (e.g., bacterial) cells, and eukaryotic cells,including, insect cells, yeast cells and mammalian cells. Viralreplication cells may comprise mammalian cells such as A549, WEHI, 313,10T1/2, BHK, MDCK, COS 1. COS 7, BSC 1, BSC 40, BMT 10, VERO. W138,HeLa, HEK293, Sams, C2C12, L cells, HT1080, HepG2 and primaryfibroblast, hepatocyte and tnyoblast cells derived from mammals. Viralreplication cells comprise cells derived from mammalian speciesincluding, but not limited to, human, monkey, mouse, rat, rabbit, andhamster or cell type, including but not limited to fibroblast,hepatocyte, tumor cell, cell line transformed cell, etc.

Mammalian Cell (Small Scale) Production of AAV Particles

Viral production disclosed herein describes processes and methods forproducing AAV particles that contact a target cell to deliver a payload,e.g. a recombinant viral construct, which comprises a polynucleotidesequence encoding a payload.

In one embodiment, the AAV particles may be produced in a viralreplication cell that comprises a mammalian cell.

Viral replication cells commonly used for production of recombinant AAVparticles include, but are not limited to 293 cells, COS cells, HeLacells, KB cells, and other mammalian cell lines as described in U.S.Pat, Nos, 6,156,303, 5,387,484, 5,741,683, 5,691,176, and 5,688,676;U.S. patent application 2002/0081721, and International PatentApplications WO 00/47757, WO 00/24916, and WO 96/17947, the contents ofeach of which are herein incorporated by reference in their entireties.

In one embodiment, AAV particles are produced in mammalian-cells whereinall three VP proteins are expressed at a stoichiometry approaching1:1:10 (VP1:VP2:VP3). The regulatory mechanisms that allow thiscontrolled level of expression include the production of two mRNAs, onefor VP1 and the other for VP2 and VP3, produced by differentialsplicing.

In another embodiment, AAV particles are produced in mammalian cellsusing a triple transfection method wherein a payload construct,parvoviral Rep and parvoviral Cap and a helper construct are comprisedwithin three different constructs. The triple transfection method of thethree components of AAV particle production may be utilized to producesmall lots of virus for assays including transduction efficiency, targettissue (tropism) evaluation, and stability.

AAV particles described herein may be produced by triple transfection orbaculovirus mediated virus production, or any other method known in theart. Any suitable permissive or packaging cell known in the art may beemployed to produce the vectors. Mammalian cells are often preferred.Also preferred are trans-complementing packaging cell lines that providefunctions deleted from a replication-defective helper virus, e.g., 293cells or other E1a trans-complementing cells.

The gene cassette may contain some or all of the parvovirus (e.g., AAV)cap and rep genes. Preferably, however, some or all of the cap and repfunctions are provided in trans by introducing a packaging vector(s)encoding the capsid and/or Rep proteins into the cell. Most preferably,the gene cassette does not encode the capsid or Rep proteins.Alternatively, a packaging cell line is used that is stably transformedto express the cap and/or rep genes.

Recombinant AAV virus particles are, in some cases, produced andpurified from culture supernatants according to the procedure asdescribed in US20160032254, the contents of which are incorporated byreference. Production may also involve methods known in the artincluding those using 293T cells, sf9 insect cells, triple transfectionor any suitable production method.

In some cases, 293T cells (adhesion/suspension) are transfected withpolyethylenimine (PEI) with plasmids required for production of AAV,i.e., AAV2 rep, an adenoviral helper construct and an ITR flankedtransgene cassette. The AAV2 rep plasmid also contains the cap sequenceof the particular virus being studied. Twenty-four hours aftertransfection (no medium changes for suspension), which occurs inDMEM/F17 with/without serum, the medium is replaced with fresh mediumwith or without serum. Three (3) days after transfection, a sample istaken from the culture medium of the 293 adherent cells. Subsequentlycells are scraped, or suspension cells are pelleted, and transferredinto a receptacle. For adhesion cells, after centrifugation to removecellular pellet, a second sample is taken from the supernatant afterscraping. Next, cell lysis is achieved by three consecutive freeze-thawcycles (−80 C to 37 C) or adding detergent triton. Cellular debris isremoved by centrifugation or depth filtration and sample 3 is taken fromthe medium. The samples are quantified for AAV particles by DNaseresistant genome titration by DNA qPCR. The total production yield fromsuch a transfection is equal to the particle concentration from sample3.

AAV particle titers are measured according to genome copy number (genomeparticles per milliliter). Genome particle concentrations are based onDNA qPCR of the vector DNA as previously reported (Clark et al. (1999)Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Then,6:272-278).

Baculovirus

Particle production disclosed herein describes processes and methods forproducing AAV particles that contact a target cell to deliver a payloadconstruct which comprises a polynucleotide sequence encoding a payload.

Briefly, the viral construct vector and the AAV payload construct vectorare each incorporated by a transposon donor/acceptor system into abactnid, also known as a baculovirus plasmid, by standard molecularbiology techniques known and performed by a person skilled in the art.Transfection of separate viral replication cell populations produces twobaculoviruses, one that comprises the viral construct expression vector,and another that comprises the AAV payload construct expression vector.The two baculoviruses may be used to infect a single viral replicationcell population for production of AAV particles.

Baculovirus expression vectors for producing viral particles in insectcells, including but not limited to Spodoptera frugipercia (Sf9) cells,provide high titers of viral particle product. Recombinant baculovirusencoding the viral construct expression vector and AAV payload constructexpression vector initiates a productive infection of viral replicatingcells. Infectious baculovirus particles released from the primaryinfection secondarily infect additional cells in the culture,exponentially infecting the entire cell culture population in a numberof infection cycles that is a function of the initial multiplicity ofinfection, see Urabe, M. et at, J Virol. 2006 Feb; 80 (4):1874-85, thecontents of which are herein incorporated by reference in theirentirety.

Production of AAV particles with baculovirus in an insect cell systemmay address known baculovirus genetic and physical instability. In oneembodiment, the production system addresses baculovirus instability overmultiple passages by utilizing a titerless infected-cells preservationand scale-up system. Small scale seed cultures of viral producing cellsare transfected with viral expression constructs encoding thestructural, non-structural, components of the viral particle.Baculovirus-infected viral producing cells are harvested into aliquotsthat may be cryopreserved in liquid nitrogen; the aliquots retainviability and infectivity for infection of large scale viral producingcell culture Wasailko D J et al., Protein Expr Purif. 2009 June;65(2):122-32, the contents of which are herein incorporated by referencein their entirety.

A genetically stable baculovirus may be used to produce source of theone or more of the components for producing AAV particles ininvertebrate cells. In one embodiment, defective baculovirus expressionvectors may be maintained episomally in insect cells. In such anembodiment the bacmid vector is engineered with replication controlelements, including but not limited to promoters, enhancers, and/orcell-cycle regulated replication elements.

In one embodiment, baculoviruses may be engineered with a (non-)selectable marker for recombination into the chitinase/cathepsin locus.The chia/v-cath locus is non-essential for propagating baculovirus intissue culture, and the V-cath (EC 3,4.22.50) is a. cysteineendoprotease that is most active on Arg-Arg dipeptide containingsubstrates, The Arg-Arg dipeptide is present in densovirus andparvovirus capsid structural proteins but infrequently occurs independovirus VP1.

In one embodiment, stable viral replication cells permissive forbaculovirus infection are engineered with at least one stable integratedcopy of any of the elements necessary for AAV replication and viralparticle production including, but not limited to, the entire AAVgenome, Rep and Cap genes, Rep genes, Cap genes, each Rep protein as aseparate transcription cassette, each VP protein as a separatetranscription cassette, the AAP (assembly activation protein), or atleast one of the baculovirus helper genes with native or non-nativepromoters.

Large-Scale Production

In some embodiments, AAV particle production may be modified to increasethe scale of production. Large scale viral production methods accordingto the present disclosure may include any of those taught in U.S. Pat.Nos. 5,756,283, 6,258,595, 6,261,551, 6,270,996, 6,281,010, 6,365,394,6,475,769 6,482,634, 6,485,966, 6,943,019, 6,953,690, 7,022,5197,238,526, 7,291,498 and 7,491,508 or International Publication Nos.WO1996039530, WO1998010088, WO1999014354, WO1999015685, WO1999047691,WO2000055342, WO2000075353 and WO2001023597, the contents of each ofwhich are herein incorporated by reference in their entirety. Methods ofincreasing viral particle production scale typically comprise increasingthe number of viral replication cells. In some embodiments, viralreplication cells comprise adherent cells. To increase the scale ofviral particle production by adherent viral replication cells, largercell culture surfaces are required. In some cases, large-scaleproduction methods comprise the use of roller bottles to increase cellculture surfaces. Other cell culture substrates with increased surfaceareas are known in the art. Examples of additional adherent cell cultureproducts with increased surface areas include, but are not limited toCELLSTACK®, CELLCUBE® (Corning Corp., Corning, N.Y.) and NUNC™ CELLFACTORY™ (Thermo Scientific, Waltham, Mass.) In some cases, large-scaleadherent cell surfaces may comprise from about 1,000 cm² to about100,000 cm². In some cases, large-scale adherent cell cultures maycomprise from about 10⁷ to about 10⁹ cells, from about 10⁸ to about 10¹⁹cells, from about 10⁹ to about 10¹² cells or at least 10¹² cells. Insome cases, large-scale adherent cultures may produce from about 10⁹ toabout 10¹², from about 10¹⁰ to about 10¹³, from about 10¹¹ to about10¹⁴, from about 10¹² to about 10¹⁵ or at least 10¹⁵ viral particles.

In some embodiments, large-scale viral production methods of the presentdisclosure may comprise the use of suspension cell cultures. Suspensioncell culture allows for significantly increased numbers of cells.Typically, the number of adherent cells that can be grown on about 10-50cm² of surface area can be grown in about 1 cm³ volume in suspension.

Transfection of replication cells in large-scale culture formats may becarried out according to any methods known in the art. For large-scaleadherent cell cultures, transfection methods may include, but are notlimited to the use of inorganic compounds (e.g. calcium phosphate),organic compounds [e.g. polyethyleneimine (PEI)] or the use ofnon-chemical methods (e.g. electroporation.) With cells grown insuspension, transfection methods may include, but are not limited to theuse of calcium phosphate and the use of PEI. In some cases, transfectionof large scale suspension cultures may be carried out according to thesection entitled “Transfection Procedure” described in Feng, L. et at.,2008. Biotechnol Appl. Biochem, 50:121-32, the contents of which areherein incorporated by reference in their entirety. According to suchembodiments, PEI-DNA complexes may be formed for introduction ofplasmids to be transfected. In some cases, cells being transfected withPEI-DNA complexes may be ‘shocked’ prior to transfection. This compriseslowering cell culture temperatures to 4° C. for a period of about 1hour. In some cases, cell cultures may be shocked for a period of fromabout 10 minutes to about 5 hours. In some cases, cell cultures may beshocked at a temperature of from about 0° C. to about 20° C.

In some cases, transfections may include one or more vectors forexpression of an RNA effector molecule to reduce expression of nucleicacids from one or more AAV payload construct. Such methods may enhancethe production of viral particles by reducing cellular resources wastedon expressing payload constructs. In some cases, such methods may becarried according to those taught in US Publication No. US2014/0099666,the contents of which are herein incorporated by reference in theirentirety.

Bioreactors

In some embodiments, cell culture bioreactors may be used for largescale viral production.. In some cases, bioreactors comprise stirredtank reactors. Such reactors generally comprise a vessel, typicallycylindrical in shape, with a stirrer (e.g. impeller.) In someembodiments, such bioreactor vessels may be placed within a water jacketto control vessel temperature and/or to minimize effects from ambienttemperature changes. Bioreactor vessel volume may range in size fromabout 500 ml to about 2 L, from about 1 L to about 5 L, from about 2.5 Lto about 20 L, from about 10 L to about 50 L, from about 25 L to about100 L, from about 75 L to about 500 L, from about 250 L to about 2,000L, from about 1,000 L to about 10,000 L, from about 5,000 L to about50,000 L or at least 50,000 L. Vessel bottoms may be rounded or flat. Insome cases, animal cell cultures may be maintained in bioreactors withrounded vessel bottoms.

In some cases, bioreactor vessels may be warmed through the use of athermocirculator. Thermocirculators pump heated water around waterjackets. In some cases, heated water may be pumped through pipes (e.g.coiled pipes) that are present within bioreactor vessels. In some cases,warm air may be circulated around bioreactors, including, but notlimited to air space directly above culture medium. Additionally, pH andCO₂ levels may be maintained to optimize cell viability.

In some cases, bioreactors may comprise hollow-fiber reactors.Hollow-fiber bioreactors may support the culture of both anchoragedependent and anchorage independent cells. Further bioreactors mayinclude, but are not limited to packed-bed or fixed-bed bioreactors.Such bioreactors may comprise vessels with glass beads for adherent cellattachment. Further packed-bed reactors may comprise ceramic beads.

In some cases, viral particles are produced through the use of adisposable bioreactor. In some embodiments, such bioreactors may includeWAVE™ disposable bioreactors.

In some embodiments, AAV particle production in animal cell bioreactorcultures may be carried out according to the methods taught in U.S. Pat.Nos. 5,064764, 6,194,191, 6,566,118, 8,137,948 or US Patent. ApplicationNo. US2011/0229971, the contents of each of which are hereinincorporated by reference in their entirety.

Cell Lysis

Cells of the disclosure, including, but not limited to viral productioncells, may be subjected to cell lysis according to any methods known inthe art. Cell lysis may be carried out to obtain one or more agents(e.g. viral particles) present within any cells of the disclosure. Insome embodiments, cell lysis may be carried out according to any of themethods listed in U.S. Pat. Nos. 7,326,555, 7,579,181, 7,048,920,6,410,300, 6,436,394, 7,732,129, 7,510,875, 7,445,930, 6,726,907,6,194,191, 7,125,706, 6,995,006, 6,676,935, 7,968,333, 5,756,283,6,258,595, 6,261,551, 6,270,996, 6,281,010, 6,365,394, 6,475,769,6,482,634, 6,485,966, 6,943,019, 6,953,690, 7,022,519, 7,238,526,7,291,498 and 7,491,508 or International Publication Nos. WO1996039530,WO1998010088, WO1999014354, WO1999015685, WO1999047691, WO2000055342,WO2000075353 and WO2001023597, the contents of each of which are hereinincorporated by reference in their entirety. Cell lysis methods may bechemical or mechanical. Chemical cell lysis typically comprisescontacting one or more cells with one or more lysis agent. Mechanicallysis typically comprises subjecting one or more cells to one or morelysis condition and/or one or more lysis force.

In some embodiments, chemical lysis may be used to lyse cells. As usedherein, the term “lysis agent” refers to any agent that may aid in thedisruption of a cell. In some cases, lysis agents are introduced insolutions, termed lysis solutions or lysis buffers. As used herein, theterm “lysis solution” refers to a solution (typically aqueous)comprising one or more lysis agent. In addition to lysis agents, lysissolutions may include one or more buffering agents, solubilizing agents,surfactants, preservatives, cryoprotectants, enzymes, enzyme inhibitorsand/or chelators. Lysis buffers are lysis solutions comprising one ormore buffering agent. Additional components of lysis solutions mayinclude one or more solubilizing agent. As used herein, the term“solubilizing agent” refers to a compound that enhances the solubilityof one or more components of a solution and/or the solubility of one ormore entities to which solutions are applied. In some cases,solubilizing agents enhance protein solubility. In some cases,solubilizing agents are selected based on their ability to enhanceprotein solubility while maintaining protein conformation and/oractivity.

Exemplary lysis agents may include any of those described in U.S. Pat.Nos. 8,685,734, 7,901,921, 7,732,129, 7,223,585, 7,125,706, 8,236,495,8,110,351, 7,419,956, 7,300,797, 6,699,706 and 6,143,567, the contentsof each of which are herein incorporated by reference in their entirety.In some cases, lysis agents may be selected from lysis salts, amphotericagents, cationic agents, ionic detergents and non-ionic detergents.Lysis salts may include, but are not limited to sodium chloride (NaCl)and potassium chloride (KCl). Further lysis salts may include any ofthose described in U.S. Pat. Nos. 8,614,101, 7,326,555, 7,579,181,7,048,920, 6,410,300, 6,436,394, 7,732,129, 7,510,875, 7,445,930,6,726,907, 6,194,191, 7,125,706, 6,995,006, 6,676,935 and 7,968,333, thecontents of each of which are herein incorporated by reference in theirentirety. Concentrations of salts may be increased or decreased toobtain an effective concentration for rupture of cell membranes.Amphoteric agents, as referred to herein, are compounds capable ofreacting as an acid or a base. Amphoteric agents may include, but arenot limited to lysophosphatidylcholine, 3-((3-Cholamidopropyl)dimethylammonium)-1-propanesulfonate (CHAPS), ZWITTERGENT® and the like.Cationic agents may include, but are not limited to,cetyltrimethylarnmonium bromide (C (16) TAB) and Benzalkonium chloride.Lysis agents comprising detergents may include ionic detergents ornon-ionic detergents. Detergents may function to break apart or dissolvecell strictures including, but not limited to cell membranes, cellwalls, lipids, carbohydrates, lipoproteins and glycoproteins. Exemplaryionic detergents include any of those taught in U.S. Pat. Nos. 7,625,570and 6,593,123 or US Publication No. US2014/0087361, the contents of eachof which are herein incorporated by reference in their entirety. Someionic detergents may include, but are not limited to sodium dodecylsulfate (SDS), cholate and deoxycholate. In some cases, ionic detergentsmay be included in lysis solutions as a solubilizing agent. Non-ionicdetergents may include, but are not limited to octylglucoside,digitonin, lubrol, C12E8, TWEEN®-20, TWEEN®-80, Triton X-100 andNoniodet P-40. Non-ionic detergents are typically weaker lysis agents,but may be included as solubilizing agents for solubilizing cellularand/or viral proteins. Further lysis agents may include enzymes andurea. In some cases, one or more lysis agents may be combined in a lysissolution in order to enhance one or more of cell lysis and proteinsolubility. In some cases, enzyme inhibitors may be included in lysissolutions in order to prevent proteolysis that may be triggered by cellmembrane disruption.

In some embodiments, mechanical cell lysis is carried out. Mechanicalcell lysis methods may include the use of one or more lysis conditionand/or one or more lysis force. As used herein, the term “lysiscondition” refers to a state or circumstance that promotes cellulardisruption. Lysis conditions may comprise certain temperatures,pressures, osmotic purity, salinity and the like. In some cases, lysisconditions comprise increased or decreased temperatures. According tosome embodiments, lysis conditions comprise changes in temperature topromote cellular disruption. Cell lysis carried out according to suchembodiments may include freeze-thaw lysis. As used herein, the term“freeze-thaw lysis” refers to cellular lysis in which a cell solution issubjected to one or more freeze-thaw cycle. According to freeze-thawlysis methods, cells in solution are frozen to induce a mechanicaldisruption of cellular membranes caused by the formation and expansionof ice crystals. Cell solutions used according freeze-thaw lysismethods, may further comprise one or more lysis agents, solubilizingagents, buffering agents, crvoprotectants, surfactants, preservatives,enzymes, enzyme inhibitors and/or chelators. Once cell solutionssubjected to freezing are thawed, such components may enhance therecovery of desired cellular products. In some cases, one or morecryoprotectants are included in cell solutions undergoing freeze-thawlysis. As used herein, the term “cryoprotectant” refers to an agent usedto protect one or more substance from damage due to freezing.Cryoprotectants may include any of those taught in US Publication No,US:2013/0323302 or U.S. Pat. Nos. 6,503,888, 6,180,613, 7,888,096,7,091,030, the contents of each of which are herein incorporated byreference in their entirety. In sonic cases, cryoprotectants mayinclude, but are not limited to dimethyl sulfoxide, 1,2-propanediol,2,3-butanediol, thrtnamide, glycerol, ethylene glycol, 1,3-propanedioland n-dimethyl formamide, polyvinylpyrrolidone, hydroxyethyl starch,agarose, dextrans, inositol, glucose, hydroxyethylstarch, lactose,sorbitol, methyl glucose, sucrose and urea. In some embodiments,freeze-thaw lysis may be carried out according to any of the methodsdescribed in U.S. Pat. No. 7,704,721, the contents of which are hereinincorporated by reference in their entirety.

As used herein, the tern “lysis force” refers to a physical activityused to disrupt a cell. Lysis forces may include, but are not limited tomechanical forces, sonic forces, gravitational forces, optical forces,electrical forces and the like. Cell lysis carried out by mechanicalforce is referred to herein as “mechanical lysis,” Mechanical forcesthat may be used according to mechanical lysis may include high shearfluid forces. According to such methods of mechanical lysis, amicrotluidizer may be used. Microfluidizers typically comprise an inletreservoir where cell solutions may be applied. Cell solutions may thenbe pumped into an interaction chamber via a pump (e.g. high-pressurepump) at high speed and/or pressure to produce shear fluid forces.Resulting lysates may then be collected in one or more output reservoir.Pump speed and/or pressure may be adjusted to modulate cell lysis andenhance recovery of products (e.g. viral particles.) Other mechanicallysis methods may include physical disruption of cells by scraping.

Cell lysis methods may be selected based on the cell culture format ofcells to be lysed. For example, with adherent cell cultures, somechemical and mechanical lysis methods may be used. Such mechanical lysismethods may include freeze-thaw lysis or scraping. In another example,chemical lysis of adherent cell cultures may be carried out throughincubation with lysis solutions comprising surfactant, such asTriton-X-100. In some cases, cell lysates generated from adherent cellcultures may be treated with one more nuclease to lower the viscosity ofthe lysates caused by liberated DNA.

In one embodiment, a method for harvesting AAV particles without lysismay be used for efficient and scalable AAV particle production. In anon-limiting example, AAV particles may be produced by culturing an AAVparticle lacking a heparin binding site, thereby allowing the AAVparticle to pass into the supernatant, in a cell culture, collectingsupernatant from the culture; and isolating the AAV particle from thesupernatant, as described in US Patent Application 20090275107, thecontents of which are incorporated herein by reference in theirentirety.

Clarification

Cell lysates comprising viral particles may be subjected toclarification. Clarification refers to initial steps taken inpurification of viral particles from cell lysates. Clarification servesto prepare lysates for further purification by removing larger,insoluble debris. Clarification steps may include, but are not limitedto centrifugation and filtration. During clarification, centrifugationmay be carried out at low speeds to remove larger debris only.Similarly, filtration may be carried out using filters with larger poresizes so that only larger debris is removed. In some cases, tangentialflow filtration may be used during clarification. Objectives of viralclarification include high throughput processing of cell lysates and tooptimize ultimate viral recovery. Advantages of including aclarification step include scalability for processing of larger volumesof lysate. In some embodiments, clarification may be carried outaccording to any of the methods presented in U.S. Pat. Nos. 8,524,446,5,756,283, 6,258,595, 6,261,551, 6,270,996, 6,281,010, 6,365,394,6,475,769, 6,482,634, 6,485,966, 6,943,019, 6,953,690, 7,022,519,7,238,526, 7,291,498, 7,491,508, US Publication Nos. US2013/0045186,U52011/0263027, US2011/0151434, US2003/0138772, and InternationalPublication Nos. WO2002012455, WO1996039530, WO1998010088, WO1999014354,WO1999015685, WO1999047691, WO2000055342, WO2000075353 and WO2001023597,the contents of each of which are herein incorporated by reference intheir entirety.

Methods of cell lysate clarification by filtration are well understoodin the art and may be carried out according to a variety of availablemethods including, but not limited to passive filtration and flowfiltration. Filters used may comprise a variety of materials and poresizes. For example, cell lysate filters may comprise pore sizes of fromabout 1 μM to about 5 μM, from about 0.5 μM to about 2 μM, from about0.1 μM to about 1 μM, from about 0.05 μM to about 0.05 μM. and fromabout 0.001 μM to about 0.1 μM. Exemplary pore sizes for cell lysatefilters may include, but are not limited to, 2.0, 1.9, 1.8, 1.7, 1.6,1.5, 1.4, 1.3, 1.2, 1.1, 1, 0,9, 0,8, 0,7, 0,6, 0,5, 0,4, 0,3, 0,2, 0,1,0,95, 0,9, 0,85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35,0.3, 0.25, 0.2, 0.15, 0.1, 0.05, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17,0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05,0.04, 0.03, 0.02, 0.01, 0.02, 0.019, 0.018, 0.017, 0,016, 0.015, 0.014,0,013, 0.012, 0.011, 0.01, 0.009, 0.008, 0.007, 0.006, 0.005, 0.004,0.003, 0.002, 0.001 and 0.001 μM. In one embodiment, clarification maycomprise filtration through a filter with 2.0 μM. pore size to removelarge debris, followed by passage through a filter with 0.45 μM poresize to remove intact cells.

Filter materials may be composed of a variety of materials. Suchmaterials may include, but are not limited to polymeric materials andmetal materials (e.g. sintered metal and pored aluminum.) Exemplarymaterials may include, but are not limited to nylon, cellulose materials(e.g., cellulose acetate), polyvinylidene fluoride (PVDF),polyethersulfone, polyamide, polysulfone, polypropylene, andpolyethylene terephthalate. In some cases, filters useful forclarification of cell lysates may include, but are not limited toULTIPLEAT PROFILE™ filters (Pall Corporation, Port Washington, N.Y.),SUPOR™ membrane filters (Pall Corporation. Port Washington, N.Y.)

In some cases, flow filtration may be carried out to increase filtrationspeed and/or effectiveness. In some cases, flow filtration may comprisevacuum filtration. According to such methods, a vacuum is created on theside of the filter opposite that of cell lysate to be filtered. In somecases, cell lysates may be passed through filters by centrifugal forces.In some cases, a pump is used to force cell lysate through clarificationfilters. Flow rate of cell lysate through one or more filters may bemodulated by adjusting one of channel size and/or fluid pressure.

According to some embodiments, cell lysates may be clarified bycentrifugation. Centrifugation may be used to pellet insoluble particlesin the lysate. During clarification, centrifugation strength [expressedin terms of aravitational units (g), which represents multiples ofstandard gravitational force] may be lower than in subsequentpurification steps. In some cases, centrifugation may be carried out oncell lysates at from about 200 g to about 800 g, from about 500 g toabout 1500 g, from about 1000 g to about 5000 g, from about 1200 g toabout 10000 g or from about 8000 g to about 15000 g. In someembodiments, cell lysate centrifugation is carried out at 8000 g for 15minutes. In some cases, density gradient centrifugation may be carriedout in order to partition particulates in the cell lysate bysedimentation rate. Gradients used according to methods of the presentdisclosure may include, but are not limited to cesium chloride gradientsand iodixanol step gradients,

Purification: Chromatography

In some cases, AAV particles may be purified from clarified cell lysatesby one or more methods of chromatography. Chromatography refers to anynumber of methods known in the art for separating out one or moreelements from a mixture. Such methods may include, but are not limitedto ion exchange chromatography (e.g. cation exchange chromatography andanion exchange chromatography), immunoaffinity chromatography andsize-exclusion chromatography. In some embodiments, methods of viralchromatography may include any of those taught in U.S. Pat. Nos.5,756,283, 6,258,595, 6,261,551, 6,270,996, 6,281,010, 6,365,394,6,475,769, 6,482,634, 6,485,966, 6,943,019, 6,953,690, 7,022,519,7,238,526, 7,291,498 and 7,491,508 or International Publication Nos.WO1996039530, WO1998010088, WO1999014354, WO1999015685, WO1999047691,WO2000055342, WO2000075353 and WO2001023597, the contents of each ofwhich are herein incorporated by reference in their entirety.

In some embodiments, ion exchange chromatography may be used to isolateviral particles. Ion exchange chromatography is used to bind viralparticles based on charge-charge interactions between capsid proteinsand charged sites present on a stationary phase, typically a columnthrough which viral preparations (e.g. clarified lysates) are passed.After application of viral preparations, bound viral particles may thenbe eluted by applying an elution solution to disrupt the charge-chargeinteractions. Elution solutions may be optimized by adjusting saltconcentration and/or pH to enhance recovery of bound viral particles.Depending on the charge of viral capsids being isolated, cation or anionexchange chromatography methods may be selected. Methods of ion exchangechromatography may include, but are not limited to any of those taughtin U.S. Pat. Nos. 7,419,817, 6,143,548, 7,094,604, 6,593,123, 7,015,026and 8,137,948, the contents of each of which are herein incorporated byreference in their entirety.

In some embodiments, immunoaffinity chromatography may be used.Immunoaffinity chromatography is a form of chromatography that utilizesone or more immune compounds (e.g. antibodies or antibody-relatedstructures) to retain viral particles. Immune compounds may hindspecifically to one or more structures on viral particle surfaces,including, but not limited to one or more viral coat protein. In somecases, immune compounds may be specific for a particular viral variant.In some cases, immune compounds may bind to multiple viral variants. Insome embodiments, immune compounds may include recombinant single-chainantibodies. Such recombinant single chain antibodies may include thosedescribed in Smith, R. H. et al., 2009. Mol. Ther. 17(11):1888-96, thecontents of which are herein incorporated by reference in theirentirety. Such immune compounds are capable of binding to several AAVcapsid variants, including, but not limited to AAV1, AAV2, AAV6 andAAV8.

In some embodiments, size-exclusion chromatography (SEC) may be used.SEC may comprise the use of a gel to separate particles according tosize. in viral particle purification, SEC filtration is sometimesreferred to as “polishing.” In some cases, SEC may be carried out togenerate a final product that is near-homogenous. Such final productsmay in some cases be used in pre-clinical studies and/or clinicalstudies (Kotin, R. M. 2011. Human Molecular Genetics. 20(1):R2-R6, thecontents of which are herein incorporated by reference in theirentirety.) In some cases, SEC may be carried out according to any of themethods taught in U.S. Pat. Nos. 6,143,548, 7,015,026, 8,476,418,6,410,300, 8,476,418, 7,419,817, 7,094,604, 6,593,123, and 8,137,948,the contents of each of which are herein incorporated by reference intheir entirety.

In one embodiment, the compositions comprising at least one AAV particlemay be isolated or purified using the methods described in U.S. Pat. No.6,146,874, the contents of which are herein incorporated by reference inits entirety.

In one embodiment, the compositions comprising at least one AAV particlemay be isolated or purified using the methods described in U.S. Pat. No.6,660,514, the contents of which are herein incorporated by reference inits entirety.

In one embodiment, the compositions comprising at least one AAV particlemay be isolated or purified using the methods described in U.S. Pat. No.8,283,151, the contents of which are herein incorporated by reference inits entirety.

In one embodiment, the compositions comprising at least one AAV particlemay be isolated or purified using the methods described in U.S. Pat. No.8,524,446, the contents of which are herein incorporated by reference inits entirety. introduction into cells

To ensure the chemical and biological stability of siRNA duplexes, it isimportant to deliver polynucleotides encoding the siRNAs inside thetarget cells. The polynucleotides of the present disclosure may beintroduced into cells using any of a variety of approaches.

In some embodiments, the polynucleotide of the present disclosure isintroduced into a cell by contacting the cell with the polynucleotide.In some embodiments, the polynucleotide is introduced into a cell bycontacting the cell with a composition comprising the polynucleotide anda lipophilic carrier. In other embodiments, the pol.ynucleotide isintroduced into a cell by transfecting or infecting the cell with avector comprising nucleic acid sequences capable of producing the siRNAduplex when transcribed in the cell.

In some embodiments, the siRNA duplex is introduced into a cell byinjecting into the cell a vector comprising nucleic acid sequencescapable of producing the siRNA duplex when transcribed in the cell.

In other embodiments, the polynucleotides of the present disclosure maybe delivered into cells by electroporation (e.g. U.S. Patent PublicationNo. 20050014264; the content of which is herein incorporated byreference in its entirety).

In addition, the siRNA molecules inserted into viral vectors (e.g. AAVvectors) may be delivered into cells by viral infection. These viralvectors are engineered and optimized to facilitate the entry of siRNAmolecule into cells that are not readily amendable to transfection.Also, some synthetic viral vectors possess an ability to integrate theshRNA into the cell genome, thereby leading to stable siRNA expressionand long-term knockdown of a target gene. In this manner, viral vectorsare engineered as vehicles for specific delivery while lacking thedeleterious replication and/or integration features found in wild-typevirus.

In some embodiments, the cells may include, but are not limited to,cells of mammalian origin, cells of human origins, embryonic steincells, induced pluripotent stem cells, neural stem cells, and neuralprogenitor cells.

Pharmaceutical Compositions and Formulation

In addition to the pharmaceutical compositions, e.g., siRNA duplexes(including the encoding plasmids or expression vectors, such as viruses,e.g., AAV) to be delivered, provided herein are principally directed topharmaceutical compositions which are suitable for administration tohumans, it will be understood by the skilled artisan that suchcompositions are generally suitable for administration to any otheranimal, (.?g., to non-human animals, e.g. non-human mammals.Modification of pharmaceutical compositions suitable for administrationto humans in order to render the compositions suitable foradministration to various animals is well understood, and the ordinarilyskilled veterinary pharmacologist can design and/or perform suchmodification with merely ordinary, if any, experimentation. Subjects towhich administration of the pharmaceutical compositions is contemplatedinclude, but are not limited to, humans and/or other primates; mammals,including commercially relevant mammals such as cattle, pigs, horses,sheep, cats, dogs, mice, and/or rats; and/or birds, includingcommercially relevant birds such as poultry, chickens, ducks, geese,and/or turkeys.

In some embodiments, compositions are administered to humans, humanpatients or subjects. For the purposes of the present disclosure, thephrase “active ingredient” generally refers either to synthetic siRNAduplexes or to the viral vector carrying the siRNA duplexes, or to thesiRNA molecule delivered by a viral vector as described herein.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, dividing, shaping and/or packaging the product into a desiredsingle- or multi-dose unit.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the disclosure will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered.

The siRNA duplexes or viral vectors encoding them can be formulatedusing one or more excipients to: (1) increase stability; (2) increasecell transfection or transduction; (3) permit the sustained or delayedrelease; or (4) alter the biodistribution (e.g., target the viral vectorto specific tissues or cell types such as brain and motor neurons).

Formulations of the present disclosure can include, without limitation,saline, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes,core-shell nanoparticles, peptides, proteins, cells transfected withviral vectors (e.g., for transplantation into a subject), nanoparticlemimics and combinations thereof. Further, the viral vectors of thepresent disclosure may be formulated using self-assembled nucleic acidnanoparticles.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofassociating the active ingredient with an excipient and/or one or moreother accessory ingredients.

A pharmaceutical composition in accordance with the present disclosuremay be prepared, packaged, and/or sold in bulk, as a single unit dose,and/or as a plurality of single unit doses. As used herein, a “unitdose” refers to a discrete amount of the pharmaceutical compositioncomprising a predetermined amount of the active ingredient. The amountof the active ingredient is generally equal to the dosage of the activeingredient which would be administered to a subject and/or a convenientfraction of such a dosage such as, for example, one-half or one-third ofsuch a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the present disclosure mayvary, depending upon the identity, size, and/or condition of the subjectbeing treated and further depending upon the route by which thecomposition is to be administered. For example, the composition maycomprise between 0.1% and 99% (w/w) of the active ingredient. By way ofexample, the composition may comprise between 0.1% and 100%, e.g.,between .5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w)active ingredient.

In some embodiments, the formulations described herein may contain atleast one SOD1 targeting polynucleotide. As a non-limiting example, theformulations may contain 1, 2, 3, 4 or 5 polynucleotide that target SOD1 gene at different sites.

In some embodiments, a pharmaceutically acceptable excipient may be atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% pure. In some embodiments, an excipient is approved for use forhumans and for veterinary use. In some embodiments, an excipient may beapproved by United States Food and Drug Administration. In someembodiments, an excipient may be of pharmaceutical grade. In someembodiments, an excipient may meet the standards of the United StatesPharmacopoeia (USP), the European Pharmacopoeia (EP), the BritishPharmacopoeia, and/or the International Pharmacopoeia.

Excipients, which, as used herein, includes, but is not limited to, anyand all solvents, dispersion media, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, and the like, as suitedto the particular dosage form desired. Various excipients forformulating pharmaceutical compositions and techniques for preparing thecomposition are known in the art (see Remington: The Science andPractice of Pharmacy, 21^(st) Edition, A. R. Gcnnaro, Lippincott,Williams & Wilkins, Baltimore, Md., 2006; incorporated herein byreference in its entirety). The use of a conventional excipient mediummay be contemplated within the scope of the present disclosure, exceptinsofar as any conventional excipient medium may be incompatible with asubstance or its derivatives, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutical composition.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodiwn phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and/or combinations thereof.

In some embodiments, the formulations may comprise at least one inactiveingredient. As used herein, the term “inactive ingredient” refers to oneor more inactive agents included in formulations. in some embodiments,all, none or some of the inactive ingredients which may be used in theformulations of the present disclosure may be approved by the US Foodand Drug Administration (FDA).

Formulations of viral vectors carrying SOD1 targeting polynucleotidesdisclosed herein may include cations or anions. In one embodiment, theformulations include metal cations such as, but not limited to, Zn2+,Ca2+, Cu2+, Mg+ and combinations thereof.

As used herein, “pharmaceutically acceptable salts” refers toderivatives of the disclosed compounds wherein the parent compound ismodified by converting an existing acid or base moiety to its salt form(e.g., by reacting the free base group with a suitable organic acid).Examples of pharmaceutically acceptable salts include, but are notlimited to, mineral or organic acid salts of basic residues such asamines; alkali or organic salts of acidic residues such as carboxylicacids; and the like. Representative acid addition salts include acetate,acetic acid, adipate, alginate, ascorbate, aspartate, henzenesulfonate,benzene sulfonic acid, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemi sulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like, aswell as nontoxic ammonium, quaternary ammonium, and amine cations,including, but not limited to ammonium, tetramethylammonium,tetmethylammonium, inethylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. The pharmaceutically acceptablesalts of the present disclosure include the conventional non-toxic saltsof the parent compound formed, for example, from non-toxic inorganic ororganic acids. The pharmaceutically acceptable salts of the presentdisclosure can be synthesized from the parent compound which contains abasic or acidic moiety by conventional chemical methods. Generally, suchsalts can be prepared by reading the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, nonaqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred. Lists of suitable salts arefound in Remington's Pharmaceutical Sciences, 17^(th) ed., MackPublishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts:Properties, Selection, and Use, P. H. Stahl and C. G. Wermuth (eds.),Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science,66, 1-19 (1977); the content of each of which is incorporated herein byreference in their entirety.

The term “pharmaceutically acceptable solvate,” as used herein, means acompound of the disclosure wherein molecules of a suitable solvent areincorporated in the crystal lattice, A suitable solvent isphysiologically tolerable at the dosage administered. For example,solvates may be prepared by crystallization, recrystallization, orprecipitation from a solution that includes organic solvents, water, ora mixture thereof. Examples of suitable solvents are ethanol, water (forexample, mono-, di-, and tri-hydrates), N-methylpyrrolidinone (NMP),dimethyl sulfoxide (DMSO), N,Ar-dimethylformamide (DMF),N,N′-ditnethylacetarnide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, and the like. When water is the solvent, the solvate isreferred to as a “hydrate.”

According to the present disclosure, the SOD1 targeting polynucleotides,or AAV vectors comprising the same, may be formulated for CNS delivery.Agents that cross the brain blood barrier may be used. For example, somecell penetrating peptides that can target siRNA molecules to the brainblood barrier endothelium may be used to formulate the siRNA duplexestargeting SOD1 gene (e.g., Mathupala, Expert Opin Ther Pat., 2009, 19,137-140; the content of Which is incorporated herein by reference in itsentirety).

In one embodiment, the AAV particles of the disclosure may be formulatedin PBS, in combination with an ethylene oxide/propylene oxide copolymer(also known as pluronic or poloxamer).

In one embodiment, the AAV particles of the disclosure may be formulatedin PBS with 0.001% pluronic acid (F-68) (poloxamer 188) at a pH of about7.0.

In one embodiment, the AAV particles of the disclosure may be formulatedin PBS with 0.001% pluronic acid (F-68) (poloxamer 188) at a pH of about7.3.

In one embodiment, the AAV particles of the disclosure may be formulatedin PBS with 0.001% pluronic acid (F-68) (poloxamer 188) at a pH of about7.4.

In one embodiment, the AAV particles of the disclosure may be formulatedin a solution comprising sodium chloride, sodium phosphate and anethylene oxide/propylene oxide copolymer.

In one embodiment, the AAV particles of the disclosure may be formulatedin a solution comprising sodium chloride, sodium phosphate dibasi.c,sodium phosphate monobasic and poloxamerl88/pluronic acid (F-68).

Administration

The SOD1 targeting polynucleotides of the present disclosure may beadministered by any route which results in a therapeutically effectiveoutcome. These include, but are not limited to intraparenchymal (intobrain tissue), intraparenchymal (spinal cord), intraparenchymal (CNS),enteral (into the intestine), gastroenteral, epidural (into the duramatter), oral (by way of the mouth), transdermal, peridural,intracerebral (into the cerebrum), intracerebroventricular (into thecerebral ventricles), epicutaneous (application onto the skin),intradermal (into the skin itself), subcutaneous (under the skin), nasaladministration (through the nose), intravenous (into a vein),intravenous bolus, intravenous drip, intraarterial (into an artery),intramuscular (into a muscle), intracardiac (into the heart),intraosseous infusion (into the bone marrow), intrathecal (into thespinal canal), intraperitoneal, (infusion or injection into theperitoneum), intravesical infusion, intravitreal, (through the eye),intracavernous injection (into a pathologic cavity) intracavitary (intothe base of the penis), intravaainal administration, intrauterine,extra-amniotic administration, transdermal (diffusion through the intactskin for systemic distribution), transmucosal (diffusion through amucous membrane), transvaginal, insufflation (snorting), sublingual,sublabial, enema, eye drops (onto the conjunctiva), in ear drops,auricular (in or by way of the ear), buccal (directed toward the cheek),conjunctival, cutaneous, dental (to a tooth or teeth), electro-osmosis,endocervical, endositmsial, endotracheal, extracorporeal, hemodialysis,infiltration, interstitial, intra-abdominal, intra-amniotic,intra-articular, intrabiliary, intrabronchial, intrabursal,intracartilaginous (within a cartilage), intracaudal (within the caudaequine), intracisternal (within the cisterna magna cerebellomedularis),intracomeal (within the cornea), dental intracornal, intracoronary(within the coronary arteries), intracorporus cavernosum (within thedilatable spaces of the corporus cavernosa of the penis), intradiscal(within a disc), intraductal (within a duct of a gland), intraduodenal(within the duodenum), intradural (within or beneath the dmintraepidermal (to the epidermis), intraesophageal (to the esophagus),intragastric (within the stomach), intragingival (within the gingivaeintraileal (within the distal portion of the small intestine),intralesional (within or introduced directly to a localized lesion),intraluminal (within a lumen of a tube), intralymphatic (within thelymph), intramedullary (within the marrow cavity of a bone),intrameninaeal (within the meninges), intraocular (within the eve),intraovarian (within the ovary), intrapericardial (within thepericardium), intrapleural (within the pleura), intraprostatic (withinthe prostate gland), intrapulmonary (within the lungs or its bronchi),intrasinal (within the nasal or periorbital sinuses), intraspinal(within the vertebral column), intrasynovial (within the synovial cavityof a joint), intratendinous vithin a tendon), intratesticular(within thetesticle), intrathecal (within the cerebrospinal fluid at any level ofthe cerebrospinal axis), intrathoracic (within the thorax), intratubular(within the tubules of an organ), intratumor (within a tumor),intratympanic (within the auras media), intravascular (within a vesselor vessels), intraventricular (within a ventricle), iontophoresis (bymeans of electric current where ions of soluble salts migrate into thetissues of the body), irrigation (to bathe or flush open wounds or bodycavities), laryngeal (directly upon the larynx), nasogastric (throughthe nose and into the stomach), occlusive dressing technique (topicalroute administration which is then covered by a dressing which occludesthe area), ophthalmic (to the external eye), oropharyngeal (directly tothe mouth and pharynx), parenteral, percutaneous, periarticular,peridural, perineural, periodontal, rectal, respiratory (within therespiratory tract by inhaling orally or nasally for local or systemiceffect), retrobulbar (behind the pons or behind the eyeball), softtissue, subarachnoid, subconjunctival, submucosal, topical,transplacental (through or across the placenta), transtracheal (throughthe wall of the trachea), transtympanic (across or through the tympaniccavity), ureteral (to the ureter), urethral (to the urethra), vaginal,caudal block, diagnostic, nerve block, biliary perfusion, cardiacperfusion, photopheresis, intrastriatal (within the striatum) infusionor spinal.

In specific embodiments, compositions including AAV vectors comprisingat least one SOD1 targeting polynucleotide may be administered in a waywhich allows them to enter the central nervous system and penetrate intomotor neurons.

In some embodiments, the therapeutics of the present disclosure may beadministered by muscular injection. Rizvanov et al. demonstrated for thefirst time that siRNA molecules, targeting mutant human SOD1 mRNA, istaken up by the sciatic nerve, retrogradely transported to the perikaryaof motor neurons, and inhibits mutant SOD1 mRNA in SOD1^(G93A)transgenic ALS mice (Rizvanov A A et al., Exp. Brain Res., 2009, 195(1),1-4: the content of which is incorporated herein by reference in itsentirety). Another study also demonstrated that muscle delivery of AAVexpressing small hairpin RNAs (shRNAs) against the mutant SOD1 gene, ledto significant mutant SOD1 knockdown in the muscle as well asinnervating motor neurons (Towne C et al., Mol Ther., 2011; 19(2):274-283; the content of which is incorporated herein by reference in itsentirety).

In some embodiments. AAV vectors that express siRNA duplexes of thepresent disclosure may be administered to a subject by peripheralinjections and/or intranasal delivery. It was disclosed in the art thatthe peripheral administration of AAV vectors for siRNA duplexes can betransported to the central nervous system, for example, to the motorneurons (e.g., U. S. Patent Publication Nos. 20100240739; and20100130594; the content of each of which is incorporated herein byreference in their entirety).

In other embodiments, compositions comprising at least one siRNA duplexof the disclosure may be administered to a subject by intracranialdelivery (See, e.g., U. S. Pat. No. 8,119,611; the content of which isincorporated herein by reference in its entirety).

The SOD1 targeting polynucleotides of the present disclosure may beadministered in any suitable forms, either as a liquid solution orsuspension, as a solid form suitable for liquid solution or suspensionin a liquid solution. They may be formulated with any appropriate andpharmaceutically acceptable excipient.

The SOD1 targeting polynucleotides of the present disclosure may beadministered in a “therapeutically effective” amount, i.e., an amountthat is sufficient to alleviate and/or prevent at least one symptomassociated with the disease, or provide improvement in the condition ofthe subject.

In some embodiments, the pharmaceutical compositions of the presentdisclosure may be administered by intraparenchymal injection orinfusion. As used herein, “injection” and “infusion” may be usedinterchangeably and indicate the same. As a non-limiting example, thepharmaceutical compositions of the present disclosure may beadministered to a subject by intraparenchymal injection. In oneembodiment, the intraparenchymal injection may be a spinalintraparenchymal injection, wherein the pharmaceutical compositions maybe administered directly to the tissue of the spinal cord. In oneembodiment, the intraparenchymal injection may be a CNS (central nervoussystem) intraparenchymal injection wherein the pharmaceuticalcompositions may be administered directly to the tissue of the CNS.

In one embodiment, the pharmaceutical compositions of the presentdisclosure may be administered to the cisterna magna in atherapeutically effective amount to transduce spinal cord motor neuronsand/or astrocytes. As a non-limiting example, the pharmaceuticalcompositions of the present disclosure may be administeredintraparenchymal injection.

In one embodiment, the pharmaceutical compositions of the presentdisclosure may be administered by intrastriatal infusion.

In some embodiments, the pharmaceutical compositions of the presentdisclosure may be administered by intraparenchymal injection as well asby another route of administration described herein.

In some embodiments, the pharmaceutical compositions of the presentdisclosure may be administered by intraparenchymal injection to the CNS,the brain and/or the spinal cord.

In some embodiments, the pharmaceutical compositions of the presentdisclosure of the present disclosure may be administered byintraparenchymal injection and intrathecal injection. As a non-limitingexample, the pharmaceutical compositions of the present disclosure maybe administered via intraparenchymal injection and intrastriatalinjection.

In one embodiment, the AAV particle described herein is administeredvia. intraparenchymal (IPa) infusion at any level of the spinal cord, ata single or at multiple sites, at a volume of more than 1 uL. In oneembodiment, a volume of 1 uL-100 uL is administered. In one embodiment,a volume of 1 uL-240 uL is administered. In one embodiment, a volume of1 uL-240 uL is administered. In one embodiment, a volume of 1 uL-220 uLis administered. In one embodiment, a volume of between 1 uL-200 uL isadministered. In one embodiment, a volume of 1 uL-180 uL isadministered. In one embodiment, a volume of 1 uL-160 uL isadministered. In one embodiment, a volume of 1 uL-150 uL isadministered. In one embodiment, a volume of 1 uL-140 uL isadministered. In one embodiment, a volume of 1 uL-130 uL isadministered. In one embodiment, a volume of 1 uL-120 uL isadministered. In one embodiment, a volume of 1 uL-110 uL isadministered. in one embodiment, a volume of tut, 90 uL is administered.In one embodiment, a volume of between 1 uL-80 uL is administered. Inone embodiment, a volume of 1 uL-70 uL is administered. In oneembodiment, a volume of 1 uL-60 uL is administered. In one embodiment, avolume of 1 uL-50 uL is administered. In one embodiment, a volume of 1uL-40 uL is administered. In one embodiment, a volume of 1 uL-30 uL isadministered. In one embodiment, a volume of 1 uL-20 uL is administered.In one embodiment, a volume of 50 uL-60 uL is administered. In oneembodiment, a volume of 5 uL-240 uL is administered. In one embodiment,a volume of 10 uL-20 uL is administered. In one embodiment, a volume of10 uL-30 uL is administered. In one embodiment, a volume of 10 uL-40 uLis administered. In one embodiment, a volume of 10 uL-50 uL isadministered. In one embodiment, a volume of 10 uL-60 uL isadministered. In one embodiment, a volume of 10 uL-80 uL isadministered. In one embodiment, a volume of 10 uL-90 uL isadministered. In one embodiment, a volume of 20 uL-240 uL isadministered. In one embodiment, a volume of 20 uL-200 uL isadministered. In one embodiment, a volume of 20 uL-180 uL isadministered. In one embodiment, a volume of 20 uL-150 uL isadministered. In one embodiment, a volume of 20 uL-120 uL isadministered. In one embodiment, a volume of 20 uL-100 uL isadministered. In one embodiment, a volume of 20 uL-80 uL isadministered. In one embodiment, a volume of 20 uL-60 uL isadministered. In one embodiment, a volume of 20 uL-50 uL isadministered. In one embodiment, a volume of 20 uL-40 uL isadministered. In one embodiment, a volume of 20 uL-30 uL isadministered. In one embodiment, a volume of 50 uL-200 uL isadministered. In one embodiment, a volume of 50 uL-180 uL isadministered. In one embodiment, a volume of 50 uL-150 uL isadministered. In one embodiment, a volume of 50 uL-100 uL isadministered. In one embodiment, a volume of 50 uL-80 uL isadministered. In one embodiment, a volume of 50 uL-70 uL isadministered. In one embodiment, a volume of 100 uL-240 uL isadministered. In one embodiment, a volume of 100 uL-200 uL isadministered. In one embodiment, a volume of 100 uL-180 uL isadministered. In one embodiment, a volume of 100 uL-150 uL isadministered.

The spinal cord is situated within the spine. The spine consists of aseries of vertebral segments. There are 7 cervical (C1-C7), 12 thoracic(T1-T12), 5 lumbar (L1-L5), and 5 sacral (S1-S5) vertebral segments.Intraparenchymal injection or infusion into the spinal cord of AAVparticles described herein may occur at one or multiple of thesevertebral segments. For example, intraparenchymal injection or infusioninto the spinal cord of AAV particles described herein may occur at 1,2, 3, 4, 5, or more than 5 sites. The intraparenchymal injection orinfusion sites may be at one or more regions independently selected fromthe cervical spinal cord, the thoracic spinal cord, the lumbar spinalcord, and the sacral spinal cord. In sonic embodiments, AAV particlesdescribed herein are administered via intraparenchymal (IPa) infusion attwo sites into the spinal cord.

In some embodiments, the AAV particle described herein may beadministered via intraparenchymal (IPa) infusion to one or more sites(e.g., 2, 3, 4 or 5 sites) selected from C1, C2, C3, C4, C5, C6, and C7.In some embodiments, the AAV particle described herein may beadministered via intraparenchymal (IPa.) infusion to two sites selectedfrom C1, C2, C3, C4, C5, C6, and C7.

In some embodiments, the AAV particle described herein may beadministered via intraparenchymal (IPa) infusion to one or more sites(e.g., 2, 3, 4 or 5 sites) selected from T1, T2, T3, T4, T5, T6, T7, T8,T9, T10, T11, and T12. In some embodiments, the AAV particle describedherein may be administered via intraparenchymal (IPa) infusion to twosites selected from T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, andT12

In some embodiments, the AAV particle described herein may beadministered via intraparenchymal (IPa) infusion to one or more sites(e.g., 2, 3, 4 or 5 sites) selected from L1, L2, L3, L4, and L5. In someembodiments, the AAV particle described herein may be administered viaintraparenchymal (IPa) infusion to two sites selected from L1, L2, L3,L4, and L5.

In some embodiments, the AAV particle described herein may beadministered via intraparenchymal (IPa) infusion to one or more sites(e.g., 2, 3, 4 or 5 sites) selected from S1, S2, S3, S4, and S5. In someembodiments, the AAV particle described herein may he administered viaintraparenchymal (IPa) infusion to two sites selected from S1, S2, S3,S4, and S5.

In some embodiments, the AAV particle described herein may beadministered via intraparenchymal (IPa) infusion at one or more sites(e.g., 2, 3, 4 or 5 sites) selected from C1, C2, C3, C4, C5, C6, C7, T1,T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, L1, L2, L3, L4, L5, S1,S2, S3, S4, and S5. In one embodiment, the AAV particle described hereinmay be administered via intraparenchymal (IPa) infusion at two sitesselected from C1, C2, C3, C4, C5, C6, C7, T1, T2, T3, T4, T5, T6, T7,T8, T9, T10, T11, T12, L1, L2, L3, L4, L5, S1, S2, S3, S4, and S5.

In some embodiments, the AAV particle described herein may beadministered to one or more sites (e.g., 2, 3, 4 or 5 sites) selectedfrom C1, C2, C3, C4, C5, C6, C7, T1, T2, T3, T4, T5, T6, T7, T8, T9,T10, T 11, T12, L1, L2, L3, L4, and L5. In one embodiment, the AAVparticle described herein may be administered via intraparenchymal (IPa)infusion at two sites selected from C1, C2, C3, C4, C5, C6, C7, T1, 12,T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, L1, L2, L3, L4, and L5.

In some embodiments, the AAV particle described herein may beadministered to one or more levels (e.g., 2, 3, or 4 sites) selectedfrom C1, C2, C3, C4, C5, C6, C7, T1, T2, T3, T4, T5, T6, T7, T8, T9,T10, T11, and T12. In one embodiment, the AAV particle described hereinmay be administered via intraparenchymal (IPa) infusion at two sitesselected from C1, C2, C3, C4, C5, C6, C7, T1, T2, T3, T4, T5, T6, T7,T8, T9, T10, T11, and T12. As a non-limiting example, the two sites mayinclude one site from the cervical spinal cord region (e.g., C1-C7) andone site from the thoracic spinal cord region (e.g., T1-T12).

In some embodiments, the AAV particle described herein may beadministered to one or more levels (e.g.. 2, 3, or 4 sites) selectedfrom C1, C2, C3, C4, C5, C6, C7, L1, L2, L3, L4, and L5. In oneembodiment, the AAV particle described herein may be administered viaintraparenchymal (IPa) infusion at two sites selected from C1, C2, C3,C4, C5, C6, C7, L1, L2, L3, L4, and L5. As a non-limiting example, thetwo sites may include one site from the cervical spinal cord region(e.g., C1-C7) and one site from the lumbar spinal cord region (e.g.,L1-L5).

In some embodiments, the AAV particle described herein may beadministered to one or more levels (e.g., 2, 3, or 4 sites) selectedfrom T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, L1, L2, L3, L4,and L5. In one embodiment, the AAV particle described herein may beadministered via intraparenchymal (IPa) infusion at two sites selectedfrom T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, L1, L2, L3, L4,and L5. As a non-limiting example, the two sites may include one sitefrom the thoracic spinal cord region (e.g., T1-T12) and one site fromthe lumbar spinal cord region (e.g., L1-L5).

In one embodiment, the AAV particle described herein is administered viaintraparenchymal (IPa) infusion at C1, C2, C3, C4, C5, C6, C7, and/orL1.

In one embodiment, the AAV particle described herein is administered viaintraparenchymal (IPa) infusion at C1. In one embodiment, the AAVparticle described herein is administered via intraparenchymal (IPa)infusion at C2. In one embodiment, the AAV particle described herein isadministered via intraparenchymal (IPa.) infusion at C3. In oneembodiment, the AAV particle described herein is administered viaintraparenchymal (IPa) infusion at C4. In one embodiment, the AAVparticle described herein is administered via intraparenchymal (IPa)infusion at C5. In one embodiment, the AAV particle described herein isadministered via intraparenchymal (IPa) infusion at C2. In oneembodiment, the AAV particle described herein is administered viaintraparenchymal (IPa) infusion at C7.

In one embodiment, the AAV particle described herein is administered viaintraparenchymal (IPa) infusion at two sites. In one embodiment, the AAVparticle described herein is administered via intraparenchymal (IPa)infusion at C1 and C2. In one embodiment, the AAV particle describedherein is administered via intraparenchymal (IPa) infusion at C1 and C3.In one embodiment, the AAV particle described herein is administered viaintraparenchymal (IPa) infusion at C1 and C4. In one embodiment, the AAVparticle described herein is administered via intraparenchymal (IPa)infusion at C1 and C5. In one embodiment, the AAV particle describedherein is administered via intraparenchymal (IPa) infusion at C1 and C6.In one embodiment, the AAV particle described herein is administered viaintraparenchymal (IPa) infusion at C1 and C7.

In one embodiment, the AAV particle described herein is administered viaintraparenchymal (IPa) infusion at two sites, In one embodiment, the AAVparticle described herein is administered via intraparenchymal (IPa)infusion at C2 and C3. In one embodiment, the AAV particle describedherein is administered via intraparenchymal (IPa) infusion at C2 and C4.In one embodiment, the AAV particle described herein is administered viaintraparenchymal (IPa) infusion at C2 and C1. In one embodiment, the AAVparticle described herein is administered via intraparenchymal (IPa)infusion at C2 and C6. In one embodiment, the AAV particle describedherein is administered via intraparenchymal (IPa) infusion at C2 and C7.

In one embodiment, the AAV particle described herein is administered viaintraparenchymal (IPa) infusion at two sites. In one embodiment, the AAVparticle described herein is administered via intraparenchymal (IPa)infusion at C3 and C4. In one embodiment, the AAV particle describedherein is administered via intraparenchymal (IPa) infusion at C3 and C1.In one embodiment, the AAV particle described herein is administered viaintraparenchymal (IPa) infusion at C3 and C6. In one embodiment, the AAVparticle described herein is administered via intraparenchymal (IPa)infusion at C3 and C7.

In one embodiment, the AAV particle described herein is administered viaintraparenchymal (IPa) infusion at two sites. In one embodiment, the AAVparticle described herein is administered via intraparenchymal (IPa)infusion at C4 and C5. In one embodiment, the AAV particle describedherein is administered via intraparenchymal (IPa) infusion at C4 and C6.In one embodiment, the AAV particle described herein is administered viaintraparenchymal (IPa) infusion at C4 and C7.

In one embodiment, the AAV particle described herein is administered viaintraparenchymal (IPa) infusion at two sites. In one embodiment, the AAVparticle described herein is administered via intraparenchymal (IPa)infusion at C5 and C6. In one embodiment, the AAV particle describedherein is administered via intraparenchymal (IPa) infusion at C5 and C7.

In one embodiment, the AAV particle described herein is administered viaintraparenchymal (IPa) infusion at two sites. In one embodiment, the AAVparticle described herein is administered via intraparenchymal (IPa)infusion at C6 and C7 of the spinal cord.

In one embodiment, the AAV particle described herein is administered viaspinal cord infusion at two sites. In another embodiment, the AAVparticle described herein comprises administration at level C3 or C5 ofthe spinal cord. In yet another embodiment, the AAV particle describedherein are administered at levels C3 and C5 of the spinal cord,

The intraparenchymal (IPa) infusion may be for 1, 2, 3, 4, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more than60 minutes. As a non-limiting example, the infusion is for 10 minutes.As a non-limiting example, the infusion is for 11 minutes. As anon-limiting example, the infusion is for 12 minutes. As a non-limitingexample, the infusion is for 13 minutes. As a non-limiting example, theinfusion is for 14 minutes. As a non-limiting example, the infusion isfor 15 minutes.

The intraparenchymal (IPa), e.g., spinal cord, infusion may be,independently, a dose volume of about 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 80, 120, 240 or more than 240 uL. As a non-limiting example,the dose volume is about 20 uL. As a non-limiting example, the dosevolume is about 25 uL. As a non-limiting example, the dose volume isabout 30 uL. As a non-limiting example, the dose volume is about 35 uL.As a non-limiting example, the dose volume is about 40 uL. As anon-limiting example, the dose volume is about 45 uL. As a non-limitingexample, the dose volume is about 50 uL. As a non-limiting example, thedose volume is about 60 uL. As a non-limiting example, the dose volumeis about 80 uL. As a non-limiting example, the dose volume is about 120uL. As a non-limiting example, the dose volume is about 240 uL.

In one embodiment, the dose volume is 5 uL-60 uL per site ofadministration. In another embodiment, the dose volume is 25 uL-40 uLper site of administration. In one embodiment, the dose volume is 5uL-60 uL for administration to level C3, C5, C6, or C7 of the spinalcord. In one embodiment, the dose volume is 5 uL-60 uL foradministration to level C3 of the spinal cord. In another embodiment,the dose volume is 5 uL-60 uL for administration to level C5 of thespinal cord. In yet another embodiment, the dose volume is 5 uL-60 uLfor administration to level C3 of the spinal cord and the dose volumefor administration to level C5 of the spinal cord is 5 uL-60 uL. In oneembodiment, the dose volume is 256L-406L for administration to level C3,C5, C6, or C7 of the spinal cord. In one embodiment, the dose volume is25 uL-40 uL for administration to level C3 of the spinal cord. Inanother embodiment, the dose volume is 25 uL-40 uL for administration tolevel C5 of the spinal cord. In yet another embodiment, the dose volumeis 25 uL-40 uL for administration to level C3 of the spinal cord and thedose volume for administration to level C5 of the spinal cord is 25uL-40 uL.

The intraparenchymal (IPa), e.g., spinal cord, infusion may be at aninjection rate of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ormore than 15 uL/min. As a non-limiting example, the injection rate is 5uL/min.

The intraparenchymal (IPa), e.g., spinal cord, infusion may be at a dosebetween about 1×10⁶ VG and about 1×10¹⁶ VG. In some embodiments,delivery may comprise a composition concentration of about 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹⁰, 2.1×10¹¹, 2.2×10¹¹,2.3×10¹¹, 2.4×10¹¹, 2.5×10¹⁰, 2.6×10¹¹, 2.7×10¹¹, 2.8×10¹⁰, 2.9×10¹¹,3×10¹¹, 4×10¹¹, 4.1×10¹¹, 4.2×10¹¹, 4.3×1.0¹⁰, 4.4×10¹¹, 4.5×10¹¹,4.6×10¹¹, 4.7×10¹¹, 4.8×10¹¹, 4.9×10¹¹, 5×1.0¹¹, 6×10¹¹, 6.1×10¹¹,6.2×10¹¹, 6.3×10¹¹, 6.4×10¹¹, 6.5×1.0¹¹, 6.6×10¹¹, 6.7×10¹¹, 6.8×10¹¹,6.9×10¹¹, 7×10¹¹, 7.1×10¹⁰, 7.2×10¹¹, 7.3×10¹¹, 7.4×10¹¹, 7.5×10¹¹,7.6×1.0¹¹, 7.7×10¹¹, 7.8×10¹¹, 7.9×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 1.1×10¹², 1.2×10¹², 1.3×10¹², 1.4×10¹², 1.5×10¹², 1.6×10¹², 1.7×10¹²,1.8×10¹², 1.9×10¹², 2×10¹¹, 3×10¹², 4×10¹², 4.1×10¹², 4.2×10¹²,4.3×10¹², 4.4×10¹², 4.5×10¹², 4.6×10¹², 4.7×10¹², 4.8×10¹², 4.9×10¹²,5×10¹², 6×10¹², 7×10¹², 8×10¹², 8.1×10¹², 8.2×10¹², 8.3×10¹², 8.4×10¹²,8.5×10¹², 8.6×10¹², 8.7×10¹², 8.8×10¹², 8.9×10¹², 9×10¹², 1×10¹³,2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³, 6×10¹³, 6.7×10¹³, 1×10¹³, 8×10¹³,9×10¹³, 1×10¹⁴, 2×10¹⁴, 3×10¹⁴, 4×10, 5×10¹⁴, 6×10¹⁴, 7×10¹⁴, 8×10¹⁴,9×10¹⁴, 1×10¹⁵, 2×10¹⁵, 3×10¹⁵, 4×10¹⁵, 5×10¹⁵, 6×10′⁵, 7×10¹⁵, 8×10¹⁵,9×10¹⁵, or 1×10¹⁶ VG. As a non-limiting example, the dose is 4.1×10¹¹VG. As a non-limiting example, the dose is 5.0×10¹¹ VG. As anon-limiting example, the dose is 5.1×1.0¹¹ VG. As a non-limitingexample, the dose is 6.6×10¹¹ VG. As anon-limiting example, the dose is8.0×10¹¹ VG. As anon-limiting example, the close is 8.1×10¹¹ VG. As anon-limiting example, the close is 1.0×10¹² VG. As a non-limitingexample, the dose is 1.1×10¹² VG. As a non-limiting example, the dose is1.2×10¹² VG. As a non-limiting example, the dose is 1.3×10¹² VG. As anon-limiting example, the dose is 1.0×10¹⁰ vg-1.0×10¹² VU. As anon-limiting example, the dose is 5.0×10¹¹ vg-8.0×10¹¹ VG.

In one embodiment, the intraparenchymal (IPa), e.g., spinal cord,infusion may be between about 1.0×10¹³ VG/ml and about 3×10¹³ VG/ml. Inanother embodiment, the intraparenchymal (IPa), e.g., spinal cord,infusion is 1.5×10¹³ VG/ml-3.0×10¹³ VG/ml. In yet another embodiment,the intraparenchymal (IPa), e.g., spinal cord, infusion is 1.8×10¹³VG/ml-2.5×10¹³ VG/ml. In one embodiment, the intraparenchymal (IPa),e.g., spinal cord, infusion is 1.8×10¹³ VG/ml, 1.85×10¹³ VG/ml, 1.9×10¹³VG/ml, 1.95×10¹³ VG/ml, 2×10¹³ VG/ml, 2.01×10¹³ VG/ml, 2.02×10¹³ VG/ml,2.03×10¹³ VG/ml, 2.04×10¹³ VG/ml, 2.05×10¹³ VG/ml, 2.06×10¹³ VG/ml,2.07×10¹³ VG/ml, 2.08×10¹³ VG/ml, 2.09×10¹³ VG/ml, or 2.10×10¹³ VG/ml.

In one embodiment, the dose volume is 5 uL-60 uL per site ofadministration and the dose is 1.0×10¹⁰ VG-1.0×10¹² VG. In oneembodiment, the dose volume is 5 uL-60 uL per site of administration andthe dose is 5.0×10¹¹ VG-8.0×10¹¹ VG. In another embodiment, the dosevolume is 25 uL-40 uL per site of administration and the dose is 1.0×10VG-1.0×10¹² VG. In another embodiment, the dose volume is 25 uL-40 uLper site of administration and the dose is 5.0×10¹¹ VG-8.0×10¹¹ VG. Inone embodiment, the dose volume is 5 uL-60 uL for administration tolevel C3, C5, C6, or C7 of the spinal cord and the dose is 1.0×10¹⁰VG-1.0×10¹² VG. In one embodiment, the dose volume is 5 uL-60 uL foradministration to level C3, C5, C6, or C7 of the spinal cord and thedose is 5.0×10¹¹ VG-8.0×10¹¹ VG. In one embodiment, the dose volume is 5uL-60 uL for administration to level C3 of the spinal cord and the doseis 1.0×10¹⁰ VG-1.0×10¹² VG. In one embodiment, the dose volume is 5uL-60 uL for administration to level C3 of the spinal cord and the doseis 5.0×10¹¹ VG-8.0×10¹¹ VG. In another embodiment, the dose volume is 5uL-60 uL for administration to level C5 of the spinal cord and the doseis 1.0×10¹⁰ VG-1.0×10¹² VG. In another embodiment, the dose volume is 5uL-60 uL for administration to level C5 of the spinal cord and the doseis 5.0×10¹¹ VG-8.0×10¹¹ VG. In yet another embodiment: i) the dosevolume is 5 uL-60 uL for administration to level C3 of the spinal cordand the dose is 1.0×10¹⁰ VG-1.0×10¹² VG, for example, 5.0×10¹¹VG-8.0×10¹¹ VU, and ii) the dose volume for administration to level C5of the spinal cord is 5 uL-60 uL and the dose is 1.0×10¹⁰ VG-1.0×10¹²VG, for example, 5.0×10¹¹ VG-8.0×10¹¹ VG. In one embodiment, the dosevolume is 25 uL-40 uL for administration to level C3, C5, C6, or C7 ofthe spinal cord and the dose is 1,0×10¹⁰ VG-1.0×10¹² VG. In oneembodiment, the dose volume is 25 uL-40 uL for administration to levelC3, C5, C6, or C7 of the spinal cord and the dose is 5.0×10¹¹VG-8.0×10¹¹ VG. In one embodiment, the dose volume is 25 uL-40 L foradministration to level C3 of the spinal cord and the dose is 1.0×10¹⁰VG-1.0×10¹² VG. In one embodiment, the dose volume is 25 uL-40 uL foradministration to level C3 of the spinal cord and the dose is 5.0×10¹¹VG-8.0×10¹¹ VG. in another embodiment, the dose volume is 25 uL-40 uLfor administration to level C5 of the spinal cord and the dose is1.0×10¹⁰ VG-1.0×10¹² VG. In another embodiment, the dose volume is 25uL-40 uL for administration to level C5 of the spinal cord and the doseis 5.0×10¹¹ VG-8.0×10¹¹ VG. In yet another embodiment, i) the dosevolume is 25 uL-40 uL for administration to level C3 of the spinal cord,and the dose is 1.0×10¹⁰ VG-1.0×10¹² VG, for example, 5.0×10¹¹VG-8.0×10¹¹ VG, and ii) the dose volume for administration to level C5of the spinal cord is 25 uL-40 uL, and the dose is 1.0×10¹⁰ VG-1.0×10¹²VG, for example, 5.0×10¹¹ VG-8.0×10¹¹ VG.

In one embodiment, the AAV particle described herein encoding siRNAmolecules may be administered via intraparenchymal (IPa) infusion at twosites. The AAV particles may be delivered at the same or differentvolume for both sites. The AAV particles may be delivered at the same ordifferent volumes for both sites. The AAV particles may be delivered atthe same or different infusion rates for both sites.

In one embodiment, the AAV particle described herein encoding siRNAmolecules may be administered via intraparenchymal (IPa) infusion at twosites. The AAV particles may be delivered at the same volume for bothsites. The AAV particles may be delivered at the same dose for bothsites. The AAV particles may be delivered at the same infusion rates forboth sites.

In one embodiment, the AAV particle described herein encoding siRNAmolecules may be administered via intraparenchymal (IPa) infusion at twosites. The AAV particles may be delivered at different volumes for bothsites. The AAV particles may be delivered at different doses for bothsites. The AAV particles may be delivered at different infusion ratesfor both sites.

In one embodiment, the AAV particle described herein encoding siRNAmolecules may be administered via intraparenchymal (IPa) infusion at twosites. The AAV particles may be delivered at the same volume for bothsites. The AAV particles may be delivered at different dose for bothsites. The AAV particles may be delivered at different nfusion rates forboth sites.

In one embodiment, the AAV particle described herein encoding siRNAmolecules may be administered via intraparenchymal (IPa) infusion at twosites, The AAV particles may be delivered at the same volume for bothsites. The AAV particles may be delivered at different dose for bothsites. The AAV particles may be delivered at the same infusion rates forboth sites.

In one embodiment, the AAV particle described herein encoding siRNAmolecules may be administered via intraparenchymal (IPa) infusion at twosites. The AAV particles may be delivered at the same volume for bothsites. The AAV particles may be delivered at the same dose for bothsites. The AAV particles may be delivered at different infusion ratesfor both sites.

In one embodiment, the AAV particle described herein encoding siRNAmolecules may be administered via intraparenchymal (IPa) infusion at twosites. The AAV particles may be delivered at different volumes for bothsites. The AAV particles may be delivered at the same dose for bothsites. The AAV particles may be delivered at the same infusion rates forboth sites.

In one embodiment, the AAV particle described herein encoding siRNAmolecules may be administered via intraparenchymal (IPa) infusion at twosites. The AAV particles may be delivered at different volume for bothsites. The AAV particles may be delivered at different dose for bothsites. The AAV particles may be delivered at the same infusion rates forboth sites.

In one embodiment, the AAV particle described herein encoding siRNAmolecules may be administered via intraparenchymal (IPa) infusion at twosites. The AAV particles may be delivered at different volumes for bothsites. The AAV particles may be delivered at the same dose for bothsites. The AAV particles may be delivered at different infusion ratesfor both sites.

In one embodiment, the AAV particle described herein encoding siRNAmolecules may be administered via intraparenchymal (IPa) infusion at C3and C5. For the infusion at C3, the volume may be 25 uL and the dose maybe 4.1×10¹¹ vg. For the infusion at C5, the volume may be 40 uL and theclose may be 6.6×10¹¹ vg. The injection rate for both infusions may be 5uL/min for about 13 minutes.

In some embodiments, IPa infusions es., spinal cord) may result indelivery of the pharmaceutical compositions (i.e., AAV particles) alongthe extent of the rostral-caudal axis of the spinal cord. In someembodiments, IPa infusions (e.g., spinal cord) yield a rostrocaudalgradient of AAV particle transmission. In some embodiments, IPainfusions (e.g., spinal cord) result in delivery of the pharmaceuticalcompositions to regions distal to the injection site. While not wishingto be bound by theory, AAV particles of the disclosure may travel thelength of the rostral caudal axis of the spinal cord subsequent to IPainfusion at a particular site. In other words, the AAV particles may notconfined to the immediate vicinity of the injection site. As anon-limiting example, the AAV particles may be transported by atrans-synaptic (across the synapse) mechanism. This trans-synapticmechanism may follow a tract or channel present along the rostral-caudalaxis of the spinal cord.

Dosing

The pharmaceutical compositions of the present disclosure may beadministered to a subject using any amount effective for preventing andtreating a SOD1 associated disorder (e.g., ALS). The exact amountrequired will vary from subject to subject, depending on the species,age, and general condition of the subject, the severity of the disease,the particular composition, its mode of administration, its mode ofactivity, and the like.

The compositions of the present disclosure are typically formulated inunit dosage form for ease of administration and uniformity of dosage. Itwill be understood, however, that the total daily usage of thecompositions of the present disclosure may be decided by the attendingphysician within the scope of sound medical judgment. The specifictherapeutically effectiveness for any particular patient will dependupon a variety of factors including the disorder being treated and theseverity of the disorder; the activity of the specific compoundemployed; the specific composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,and route of administration,; the duration of the treatment; drugs usedin combination or coincidental with the specific compound employed; andlike factors well known in the medical arts.

In some specific embodiments, the doses of AAV vectors for deliveringsiRNA duplexes of the present disclosure may be adapted dependent on thedisease condition, the subject and the treatment strategy, etc.Typically, about 10⁵, 10⁶, 10¹², 10¹³, 10¹⁴, 10¹⁵ to 10¹⁶ viral genome(unit) may be administered per dose.

The desired dosage may be delivered three times a day, two times a day,once a day, every other day, every third day, every week, every twoweeks, every three weeks, or every four weeks.

In certain embodiments, the desired dosage may be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations). When multiple administrations are employed, splitdosing regimens such as those described herein may be used. As usedherein, a “split dose” is the division of single unit dose or totaldaily dose into two or more doses, e.g., two or more administrations ofthe single unit dose. As used herein, a “single unit dose” is a dose ofany modulatory polynucleotide therapeutic administered in one dose/atone time/single route/single point of contact, i.e., singleadministration event. As used herein, a “total daily dose” is an amountgiven or prescribed in 24 hour period. It may be administered as asingle unit dose. In one embodiment, the viral vectors comprising theSOD1 targeting polynucleotides of the present disclosure arcadministered to a subject in split doses. They may be formulated inbuffer only or in a formulation described herein.

Methods of Treatment of Disorders Associated with the Spinal Cord,Including ALS

Provided in the present disclosure are methods for introducing the SOD1targeting polynucleotides described herein into cells, the methodcomprising introducing into said cells any of the polynucleotides in anamount sufficient for degradation of target SOD1 mRNA to occur. In someaspects, the cells may be stem cells, neurons such as motor neurons,muscle cells and glial cells such as astrocytes.

Described here are methods for delivering AAV particles to the spinalcord, for the treatment of disorders associated with the spinal cord,such as, but not limited to motor neuron disease (e.g., ALS). In oneembodiment, these methods result in trans-synpatic transmission.

Disclosed in the present disclosure are also methods for treating ALSassociated with abnormal SOD1 function in a subject in need oftreatment. The method optionally comprises administering to the subjecta therapeutically effective amount of a composition comprising orencoding at least one siRNA duplex targeting SOD1 gene. Said siRNAduplex will silence SOD1 gene expression and inhibit SOD1 proteinproduction, and reduce one or more symptoms of ALS in the subject suchthat ALS is therapeutically treated.

In some embodiments, the SOD1 targeting polynucleotide of the presentdisclosure or the composition comprising or encoding is administered tothe central nervous system of the subject, In other embodiments, thesiRNA. duplex of the present disclosure or the composition comprising itis administered to the muscles of the subject

In particular, the SOD1 targeting polynucleotides may be delivered intospecific types of targeted cells, including motor neurons; glial cellsincluding oligodendrocyte, astrocyte and microglia; and/or other cellssurrounding neurons such as T cells. Studies in human ALS patients andanimal SOD1 ALS model implicated that glial cells play an early role inthe dysfunction and death of ALS neurons. Normal SOD1 in thesurrounding, protective glial cells can prevent the motor neurons fromdying even though mutant SOD1 is present in motor neurons (e.g.,reviewed by Philips and Rothstein, Exp. Neurol 2014, May 22. pii:S0014-4886(14)00157-5; the content of which is incorporated herein byreference in its entirety).

In some specific embodiments, at least one siRNA duplex targeting SOD1gene used as a therapy for ALS is inserted in a viral vector, such as anAAV vector.

In some embodiments, the present composition is administered as a singletherapeutic or combination therapeutics for the treatment of ALS.

The viral vectors comprising or encoding siRNA. duplexes targeting SOD1gene may be used in combination with one or more other therapeutic,agents. By “in combination with,” it is not intended to imply that theagents must be administered at the same time and/or formulated fordelivery together, although these methods of delivery are within thescope of the present disclosure. Compositions can be administeredconcurrently with, prior to, or subsequent to, one or more other desiredtherapeutics or medical procedures. In general, each agent will beadministered at a dose and/or on a time schedule determined for thatagent.

Therapeutic agents that may be used in combination with the SOD1targeting polynucleotides of the present disclosure can be smallmolecule compounds which are antioxidants, anti-inflammatory agents,anti-apoptosis agents, calcium regulators, antiglutamatergic agents,structural protein inhibitors, and compounds involved in metal ionregulation.

Compounds used in combination for treating ALS may include, but are notlimited to, agents that reduce oxidative stress, such as free-radicalscavengers, or Radicava (edaravone), antiglutamatergic agents: Riluzole,Topiramate, Talampanel, Lamotrigine, Dextromethorphan, Gabapentin andRMPA antagonist; Anti-apoptosis agents: Minocycline, Sodiumphenylbutyrate and Arimoclomol; Anti-inflammatory agent: ganglioside,Celecoxib, Cyclosporine, Azathioprine, Cyclophosphamide, Plasmaphoresis,Glattramer acetate and thalidomide; Ceftriaxone (Berry et al., Plos One,2013, 8(4)); Beat-lactam antibiotics; Pramipexole (a dopamine agonist)(Wang et al., Amyotrophic Lateral Scler., 2008, 9(1), 50-58); Nimesulidein U.S. Patent Publication No. 20060074991; Diazoxide disclosed in U.S.Patent Publication No. 20130143873); pyrazolone derivatives disclosed inUS Patent Publication No, 20080161378; free radical scavengers thatinhibit oxidative stress-induced cell death, such as bromocriptine (US.Patent Publication No. 20110105517); phenyl carbamate compoundsdiscussed in PCT Patent Publication No. 2013100571; neuroprotectivecompounds disclosed in U.S. Pat. Nos. 6,933,310 and 8,399,514 and USPatent Publication Nos. 20110237907 and 20140038927; and glycopeptidestaught in U.S. Patent Publication No. 20070185012; the content of eachof which is incorporated herein by reference in their entirety.

Therapeutic agents that may be used in combination therapy with thesiRNA duplexes targeting SOD1 gene of the present disclosure may behormones or variants that can protect neuron loss, such asadrenocorticotropic hormone (ACTH) or fragments thereof (e.g., U.S.Patent Publication No. 20130259875); Estrogen (e.g., U.S. Pat. Nos.6,334,998 and 6,592,845); the content of each of which is incorporatedherein by reference in their entirety.

Neurotrophic factors may be used in combination therapy with the siRNAduplexes targeting SOD1 gene of the present disclosure for treating ALS.Generally, a neurotrophic factor is defined as a substance that promotessurvival, growth, differentiation, proliferation and/or maturation of aneuron, or stimulates increased activity of a neuron. In someembodiments, the present methods further comprise delivery of one ormore trophic factors into the subject in need of treatment. Trophicfactors may include, but are not limited to, IGF-I, GDNF, BDNF, CENT,VEGF, Coliyelin, Xaliproden, Thyrotrophin-releasing hormone and ADNF,and variants thereof.

In one aspect, the AAV vector comprising at least one siRNA duplextargeting SOD1 gene may be co-administered with AAV vectors expressingneurotrophic factors such as AAV-IGF-I (Vincent et al., Neurorrolecularmedicine, 2004, 6, 79-85; the content of which is incorporated herein byreference in its entirety) and AAV-GDNF (Wang et al., J. Neurosci.,2002, 22, 6920-6928; the content of which is incorporated herein byreference in its entirety).

In some embodiments, the composition of the present disclosure fortreating ALS is administered to the subject in need intravenously,intramuscularly, subcutaneously, intraperitoneally, intrathecally,intraparenchymally (CNS, brain, and/or spinal cord) and/orintraventricularly, allowing the siRNA duplexes or vectors comprisingthe siRNA duplexes to pass through one or both the blood-brain barrierand the blood spinal cord barrier. In some aspects, the method includesadministering (e.g., intraparenchymally administering,intraventricularly administering and/or intrathecally administering)directly to the central nervous system (CNS) of a subject (using, e.g.,an infusion pump and/or a delivery scaffold) a therapeutically effectiveamount of a composition comprising at least one siRNA duplex targetingSOD1 gene or AAV vectors comprising at least one siRNA duplex targetingSOD1 gene, silencing/suppressing SOD1 gene expression, and reducing oneor more symptoms of ALS in the subject such that ALS is therapeuticallytreated.

In some embodiments, the composition of the present disclosure fortreating ALS is administered to the subject in need intraparenchymally(CNS, brain, and/or spinal cord), allowing the siRNA duplexes or vectorscomprising the siRNA duplexes to pass through one or both theblood-brain barrier and the blood spinal cord barrier.

In certain aspects, the symptoms of ALS including motor neurondegeneration, muscle weakness, muscle atrophy, the stiffness of muscle,difficulty in breathing, slurred speech, fasciculation development,frontotemporal dementia and/or premature death are improved in thesubject treated. In other aspects, the composition of the presentdisclosure is applied to one or both of the brain and the spinal cord.In other aspects, one or both of muscle coordination and muscle functionare improved. In other aspects, the survival of the subject isprolonged.

Definitions

Unless stated otherwise, the following terms and phrases have themeanings described below. The definitions are not meant to be limitingin nature and serve to provide a clearer understanding of certainaspects of the present invention.

As used herein, the term “nucleic acid”, “polynucleotide” and“oligonucleotide” refer to any nucleic acid polymers composed of eitherpolydeoxyribonucleotides (containing 2-deoxy-D-ribose), orpolyribonucleotides (containing D-ribose), or any other type ofpolynucleotide which is an N glycoside of a purine or pyrimidine base,or modified purine or pyrimidine bases. There is no intended distinctionin length between the term “nucleic acid”, “polynucleotide” and“oligonucleotide”, and these terms will be used interchangeably. Theseterms refer only to the primary structure of the molecule. Thus, theseterms include double- and single-stranded DNA, as well as double- andsingle stranded RNA.

As used herein, the term “RNA” or “RNA molecule” or “ribonucleic acidmolecule” refers to a polymer of ribonucleotides; the term “DNA” or “DNAmolecule” or “deoxyribonucleic acid molecule” refers to a polymer ofdeoxyribonucleotides. DNA and RNA can be synthesized naturally, e.g., byDNA replication and transcription of DNA, respectively; or be chemicallysynthesized. DNA and RNA can be single-stranded (i.e., ssRNA or ssDNA,respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA anddsDNA, respectively). The term “mRNA” or “messenger RNA”, as usedherein, refers to a single stranded RNA that encodes the amino acidsequence of one or more polypeptide chains.

As used herein, the term “RNA interfering” or “RNAi” refers to asequence specific regulatory mechanism mediated by RNA molecules whichresults in the inhibition or interfering or “silencing” of theexpression of a corresponding protein-coding gene. RNAi has beenobserved in many types of organisms, including plants, animals andfungi. RNAi occurs in cells naturally to remove foreign RNAs (e.g.,viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNAwhich direct the degradative mechanism to other similar RNA sequences.RNAi is controlled by the RNA-induced silencing complex (RISC) and isinitiated by short/small dsRNA molecules in cell cytoplasm, where theyinteract with the catalytic RISC component argonaute. The dsRNAmolecules can be introduced into cells exogenously. Exogenous dsRNAinitiates RNAi by activating the ribonuclease protein Dicer, which bindsand cleaves dsRNAs to produce double-stranded fragments of 21-25 basepairs with a few unpaired overhang bases on each end. These short doublestranded fragments are called small interfering RNAs (siRNAs).

As used herein, the term “small/short interfering RNA” or “siRNA” refersto an RNA molecule (or RNA analog) comprising between about 5-60nucleotides (or nucleotide analogs) which is capable of directing ormediating RNAi. Preferably, a siRNA molecule comprises between about15-30 nucleotides or nucleotide analogs, more preferably between about16-25 nucleotides (or nucleotide analogs), even more preferably betweenabout 18-23 nucleotides (or nucleotide analogs), and even morepreferably between about 19-22 nucleotides (or nucleotide analogs)(e.g., 19, 20, 21 or 22 nucleotides or nucleotide analogs). The term“short” siRNA refers to a siRNA comprising 5-23 nucleotides, preferably21 nucleotides (or nucleotide analogs), for example, 19, 20, 21 or 22nucleotides. The term “long” siRNA refers to a siRNA comprising 24-60nucleotides, preferably about 24-25 nucleotides, for example, 23, 24, 25or 26 nucleotides. Short siRNAs may, in some instances, include fewerthan 19 nucleotides, e.g., 16, 17 or 18 nucleotides, or as few as 5nucleotides, provided that the shorter siRNA retains the ability tomediate RNAi. Likewise, long siRNAs may, in some instances, include morethan 26 nucleotides, e.g., 27, 28, 29. 30, 35, 40, 45, 50, 55, or even60 nucleotides, provided that the longer siRNA retains the ability tomediate RNAi or translational repression absent further processing,e.g., enzymatic processing, to a short siRNA. siRNAs can be singlestranded RNA molecules (ss-siRNAs) or double stranded RNA molecules(ds-siRNAs) comprising a sense strand and an antisense strand whichhybridized to form a duplex structure called siRNA duplex. According tothe present disclosure, recombinant AAV vectors may encode one or moreRNAi molecules such as an siRNA, shRNA, tnicroRNA or precursor thereof.

As used herein, the term “the antisense strand” or “the first strand” or“the guide strand” of a siRNA molecule refers to a strand that issubstantially complementary to a section of about 10-50 nucleotides,e.g., about 15-30, 16-25, 18-23 or 19-22 nucleotides of the mRNA of thegene targeted for silencing. The antisense strand or first strand hassequence sufficiently complementary to the desired target mRNA sequenceto direct target-specific silencing, e.g., complementarity sufficient totrigger the destruction of the desired target mRNA by the RNAi machineryor process.

As used herein, the term “the sense strand” or “the second strand” or“the passenger strand” of a siRNA molecule refers to a strand that iscomplementary to the antisense strand or first strand. The antisense andsense strands of a siRNA molecule are hybridized to form a duplexstructure. As used herein, a “siRNA duplex” includes a siRNA strandhaving sufficient complementarity to a section of about 10-50nucleotides of the mRNA of the gene targeted for silencing and a siRNAstrand having sufficient complementarity to form a duplex with the siRNAstrand. According to the present disclosure, recombinant AAV vectors mayencode a sense and/or antisense strand.

As used herein, the term “complementary” refers to the ability ofpolynucleotides to form base pairs with one another. Base pairs aretypically formed by hydrogen bonds between nucleotide units inantiparallel polynucleotide strands. Complementary polynucleotidestrands can form base pair in the Watson-Crick manner (e.g., A to T, Ato U, C to G), or in any other manner that allows for the formation ofduplexes. As persons skilled in the art are aware, when using RNA asopposed to DNA, uracil rather than thymine is the base that isconsidered to he complementary to adenosine. However, when a U isdenoted in the context of the present disclosure, the ability tosubstitute a T is implied, unless otherwise stated. Perfectcomplementarity or 100% complementarity refers to the situation in whicheach nucleotide unit of one polynucleotide strand can form hydrogen bondwith a nucleotide unit of a second polynucleotide strand. Less thanperfect complementarity refers to the situation in which some, but notall, nucleotide units of two strands can form hydrogen bond with eachother. For example, for two 20-mers, if only two base pairs on eachstrand can form hydrogen bond with each other, the polynucleotidestrands exhibit 10% complementarity. In the same example, if 18 basepairs on each strand can form hydrogen bonds with each other, thepolynucleotide strands exhibit 90% complementarity.

As used herein, “targeting” means the process of design and selection ofnucleic acid sequence that will hybridize to a target nucleic acid andinduce a desired effect.

The term “gene expression” refers to the process by which a nucleic acidsequence undergoes successful transcription and in most instancestranslation to produce a protein or peptide. For clarity, when referenceis made to measurement of “gene expression”, this should be understoodto mean that measurements may be of the nucleic acid product oftranscription, e.g., RNA or mRNA or of the amino acid product oftranslation, e.g., polypeptides or peptides. Methods of measuring theamount or levels of RNA, mRNA, polypeptides and peptides are well knownin the art.

As used herein, the term “mutation” refers to any changing of thestructure of a gene, resulting in a variant (also called “mutant”) formthat may be transmitted to subsequent generations. Mutations in a genemay be caused by the alternation of single base in DNA, or the deletion,insertion, or rearrangement of larger sections of genes or chromosomes.

As used herein, the term “vector” means any molecule or moiety whichtransports, transduces or otherwise acts as a carrier of a heterologousmolecule such as the SOD1 targeting polynucleotides of the disclosure. A“viral vector” is a vector which comprises one or more polynucleotideregions encoding or comprising a molecule of interest, e.g., atransgene, a polynucleotide encoding a polypeptide or multi-polypeptideor a modulatory nucleic acid such as small interfering RNA (siRNA).Viral vectors are commonly used to deliver genetic materials into cells.Viral vectors are often modified for specific applications. Types ofviral vectors include retroviral vectors, lentiviral vectors, adenoviralvectors and adeno-associated viral vectors.

The term “adeno-associated virus” or “AAV” or “AAV vector” as usedherein refers to any vector which comprises or derives from componentsof an adeno associated vector and is suitable to infect mammalian cells,preferably human cells. The term AAV vector typically designates an AAVtype viral particle or virion comprising a nucleic acid moleculeencoding a siRNA duplex. The AAV vector may be derived from variousserotypes, including combinations of serotypes (i.e., “pseudotyped” AAV)or from various genomes (e.g., single stranded or self-complementary).In addition, the AAV vector may be replication defective and/ortargeted.

As used herein, the phrase “inhibit expression of a gene” means to causea reduction in the amount of an expression product of the gene. Theexpression product can be an RNA molecule transcribed from the gene(e.g., an mRNA) or a polypeptide translated from an mRNA transcribedfrom the gene. Typically, a reduction in the level of an mRNA results ina reduction in the level of a polypeptide translated therefrom. Thelevel of expression may be determined using standard techniques formeasuring mRNA or protein.

As used herein, the term “in vitro” refers to events that occur in anartificial environment, e.g., in a test tube or reaction vessel, in cellculture, in a Petri dish, etc., rather than within an organism (e.g.,animal, plant, or microbe).

As used herein, the term “in vivo” refers to events that occur within anorganism (e.g., animal, plant, or microbe or cell or tissue thereof).

As used herein, the term “modified” refers to a changed state orstructure of a molecule of the disclosure. Molecules may be modified inmany ways including chemically, structurally, and functionally.

As used herein, the term “synthetic” means produced, prepared, and/ormanufactured by the hand of man. Synthesis of polynucleotides orpolypeptides or other molecules of the present disclosure may bechemical or enzymatic.

As used herein, the term “transfection” refers to methods to introduceexogenous nucleic acids into a cell. Methods of transfection include,but are not limited to, chemical methods, physical treatments andcationic lipids or mixtures. The list of agents that can be transfectedinto a cell is large and includes, but is not limited to, siRNA, senseand/or anti-sense sequences, AAV vectors or particles. DNA encoding oneor more genes and organized into an expression plasmid, proteins,protein fragments, and more.

As used herein, “off target” refers to any unintended effect on any oneor more target, gene, or cellular transcript.

As used herein, the phrase “pharmaceutically acceptable” is employedherein to refer to those compounds, materials, compositions, and/ordosage forms which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

As used herein, the term “effective amount” of an agent is that amountsufficient to effect beneficial or desired results, for example,clinical results, and, as such, an “effective amount” depends upon thecontext in which it is being applied. For example, in the context ofadministering an agent that treats ALS, an effective amount of an agentis, for example, an amount sufficient to achieve treatment, as definedherein, of ALS, as compared to the response obtained withoutadministration of the agent.

As used herein, the term “therapeutically effective amount” means anamount of an agent to be delivered (e.g., nucleic acid, drug,therapeutic agent, diagnostic agent, prophylactic agent, etc.) that issufficient, when administered to a subject suffering from or susceptibleto an infection, disease, disorder, and/or condition, to treat, improvesymptoms of, diagnose, prevent, and/or delay the onset of the infection,disease, disorder, and/or condition.

As used herein, the term “subject” or “patient” refers to any organismto which a composition in accordance with the disclosure may beadministered, e.g., for experimental, diagnostic, prophylactic, and/ortherapeutic purposes. Typical subjects include animals (e.g , mammalssuch as mice, rats, rabbits, pigs, non-human primates such aschimpanzees and other apes and monkey species, and humans) and/orplants.

As used herein, the term “preventing” or “prevention” refers to delayingor forestalling the onset, development or progression of a condition ordisease for a period of time, including weeks, months, or years.

The term “treatment” or “treating”, as used herein, refers to theapplication of one or more specific procedures used for the cure oramelioration of a disease. In certain embodiments, the specificprocedure is the administration of one or more pharmaceutical agents. Inthe context of the present disclosure, the specific procedure is theadministration of one or more siRNA duplexes or dsRNA targeting SOD1gene.

As used herein, the term “amelioration” or “ameliorating” refers to alessening of severity of at least one indicator of a condition ordisease. For example, in the context of neurodegeneration disorder,amelioration includes the reduction of neuron loss.

As used herein, the term “administering” refers to providing apharmaceutical agent or composition to a subject.

As used herein, the term“neurodegeneration” refers to a pathologic statewhich results in neural cell death. A large number of neurologicaldisorders share neurodegeneration as a common pathological state. Forexample, Alzheimer's disease, Parkinson's disease, Huntington's disease,and amyotrophic lateral sclerosis (ALS) all cause chronicneurodegeneration, which is characterized by a slow, progressive neuralcell death over a period of several years, whereas acuteneurodegeneration is characterized by a sudden onset of neural celldeath as a result of ischemia, such as stroke, or trauma, such astraumatic brain injury, or as a result of axonal transection bydemyelination or trauma caused, for example, by spinal cord injury ormultiple sclerosis. In some neurological disorders, mainly one type ofneuron cells is degenerative, for example, motor neuron degeneration inALS.

EXAMPLES Example 1 SOD1 Targeting Polynucleotide Design (siRNA)

siRNA design is carried out to identify siRNAs targeting human SOD1gene. The design uses the SOD1 transcripts from human (GenBank accessNo. NM_000454.4; SEQ ID NO: 10). cynomolgus monkey (GenBank access No.NM_001285406.1; SEQ ID NO: 11), rhesus monkey (GenBank access No.NM_001032804.1; SEQ ID NO: 11), and sus scrofa (GenBank access No.NM_001190422.1; SEQ ID NO: 13), respectively (Table 10). The siRNAduplexes are designed with 100% identity to the human SOD1 transcriptfor positions 2-18 of the antisense strand, and partial or 100% identityto the non-human SOD1 transcript for positions 2-18 of the antisensestrand. In all siRNA duplexes, position 1 of the antisense strand isengineered to a U and position 19 of the sense strand was engineered toa C, in order to unpair the duplex at this position.

TABLE 10  SOD1 gene sequences SOD1 SEQ tran- ID scripts Access No. NO.Sequence Human NM_000454.4 10 GTTTGGGGCCAGAGTGGGCGAGGCGCGGAGGTCTGGCCTASOD1 TAAAGTAGTCGCGGAGACGGGGTGCTGGTTTGCGTCGTAG cDNATCTCCTGCAGCGTCTGGGGTTTCCGTTGCAGTCCTCGGAAC (981 bp)CAGGACCTCGGCGTGGCCTAGCGAGTTATGGCGACGAAGGCCGTGTGCGTGCTGAAGGGCGACGGCCCAGTGCAGGGCATCATCAATTTCGAGCAGAAGGAAAGTAATGGACCAGTGAAGGTGTGGGGAAGCATTAAAGGACTGACTGAAGGCCTGCATGGATTCCATGTTCATGAGTTTGGAGATAATACAGCAGGCTGTACCAGTGCAGGTCCTCACTTTAATCCTCTATCCAGAAAACACGGTGGGCCAAAGGATGAAGAGAGGCATGTTGGAGACTTGGGCAATGTGACTGCTGACAAAGATGGTGTGGCCGATGTGTCTATTGAAGATTCTGTGATCTCACTCTCAGGAGACCATTGCATCATTGGCCGCACACTGGTGGTCCATGAAAAAGCAGATGACTTGGGCAAAGGTGGAAATGAAGAAAGTACAAAGACAGGAAACGCTGGAAGTCGTTTGGCTTGTGGTGTAATTGGGATCGCCCAATAAACATTCCCTTGGATGTAGTCTGAGGCCCCTTAACTCATCTGTTATCCTGCTAGCTGTAGAAATGTATCCTGATAAACATTAAACACTGTAATCTTAAAAGTGTAATTGTGTGACTTTTTCAGAGTTGCTTTAAAGTACCTGTAGTGAGAAACTGATTTATGATCACTTGGAAGATTTGTATAGTTTTATAAAACTCAGTTAAAATGTCTGTTTCAATGACCTGTATTTTGCCAGACTTAAATCACAGATGGGTATTAAACTTGTCAGAATTTCTTTGTCATTCAAGCCTGTGAATAAAAACCCTGTATGGCACTFATTATGAGGCTATTAAAAGAATCCAAATTCAAACTAA AAAAAAAAAAAAAAAA cynomolgusNM_001285406.1 11 ATGGCGATGAAGGCCGTGTGCGTGTTGAAGGGCGACAGCC SOD1CAGTGCAGGGCACCATCAATTTCGAGCAGAAGGAAAGTA cDNAATGGACCAGTGAAGGTGTGGGGAAGCATTACAGGATTGAC (465 bp)TGAAGGCCTGCATGGATTCCATGTTCATCAGTTTGGAGATAATACACAAGGCTGTACCAGFGCAGGTCCTCACTTTAATCCTCTATCCAGACAACACGGTGGGCCAAAGGATGAAGAGAGGCATGTTGGAGACCTGGGCAATGTGACTGCTGGCAAAGATGGTGTGGCCAAGGTGTCTTTCGAAGATTCTGTGATCTCGCTCTCAGGAGACCATTCCATCATTGGCCGCACATTGGTGGTCCATGAAAAAGCAGATGACTTGGGCAAAGGTGGAAATGAAGAAAGTAAAAAGACAGGAAACGCTGGAGGTCGTCTGGCT TGTGGTGTAATTGGGATCGCCCAATAArhesus NM_001032804.1 11 ATGGCGATGAAGGCCGTGTGCGTGTTGAAGGGCGACAGCC SOD1CAGTGCAGGGCACCATCAATTTCGAGCAGAAGGAAAGTA cDNAATGGACCAGTGAAGGTGTGGGGAAGCATTACAGGATTGAC (465 bp)TGAAGGCCTGCATGGATTCCATGTTCATCAGTTTGGAGATAATACACAAGGCTGTACCAGMCAGGTCCTCACTTTAATCCTCTATCCAGACAACACGGTGGGCCAAAGGATGAAGAGAGGCATGTTGGAGACCTGGGCAATGTGACTGCTGGCAAAGATGGTGTGGCCAAGGTGTCTTTCGAAGATTCTGTGATCTCGCTCTCAGGAGACCATTCCATCATTGGCCGCACATTGGTGGTCCATGAAAAAGCAGATGACTTGGGCAAAGGTGGAAATGAAGAAAGTAAAAAGACAGGAAACGCTGGAGGTCGTCTGGCT TGTGGTGTAATTGGGATCGCCCAATAASus scrofa NM_001190422.1 13 CGTCGGCGTGTACTGCGGCCTCTCCCGCTGCTTCTGGTACCSOD1 CTCCCAGCCCGGACCGGAGCGCGCCCCCGCGAGTCATGGC cDNA GACGAAGGCCGTGTGTGTGCTGAAGGGCGACGGCCCGGTG (658 bp)CAGGGCACCATCTACTTCGAGCTGAAGGGAGAGAAGACAGTGTTAGTAACGGGAACCATTAAAGGACTGGCTGAAGGTGATCATGGATTCCATGTCCATCAGTTTGGAGATAATACACAAGGCTGTACCAGTGCAGGTCCTCACTTCAATCCTGAATCCAAAAAACATGGTGGGCCAAAGGATCAAGAGAGGCACGTTGGAGACCTGGGCAATGTGACTGCTGGCAAAGATGGTGTGGCCACTGTGTACATCGAAGATTCTGTGATCGCCCTCTCGGGAGACCATTCCATCATTGGCCGCACAATGGTGGTCCATGAAAAACCAGATGACTTGGGCAGAGGTGGAAATGAAGAAAGTACAAAGACGGGAAATGCTGGAAGTCGTTTGGCCTGTGGTGTAATTGGGATCACCCAGTAAACATTCCCTCATGCCATGGTCTGAATGCCAGTAACTCATCTGTTATCTTGCTAGTTGTAGTTGTAGAAATTTAACTTGATAAACATTAAACACTGTAACCTTA AAAAAAAAAAAAAAAAA

Example 2 Intraparenchymal Delivery of AAV to Spinal Cord

Traditional routes of AAV delivery, such as intrathecal or intravenousadministration, have not yielded robust transduction of the cervical andthoracic spinal cord in large mammals so a new route of AAVdelivery—intraparenchymal injection—was evaluated for improved cervicalspinal cord transduction efficiency. Biodistribution of viral genomesand SOD1 mRNA knockdown were evaluated in the ventral horn at multiplelevels of the spinal cord, including the cervical level.

In the first experiment, three Gottingen adult (6 months of age), femalemini-pigs weighing 14-20 kg each were utilized for the study. Animalswere not pre-screened for neutralizing antibodies to AAV. A 4-5 cmlaminectomy was performed between C3 and C5, allowing for 3 cm betweeninjections. Self-complementary (sc) AAV vectors (scAAV) with ITR to ITRsequence of SEQ m NO: 9, including an H1 promoter and modulatorypolynucleotide (SEQ ID NO: 6) comprising siRNA targeting SOD1 werepackaged in AAVrh10.

Two injections of the scAAV (titer 2.03×10¹³vg/mL) were administered,for a total doselanimal of 1.3×10¹² vg. At the rostral end of thelaminectomy, i.e. at the C3 level of the spinal cord, a single 25 μL(5.1×10¹¹ vg) volume was injected into the ventral horn of the spinalcord. At the caudal end of the laminectomy, i.e. at the C5 level of thespinal cord, a single 400 μL (8.1×10¹¹ vg) volume was injected into theventral horn of the contralateral side. Both injections wereadministered at the rate of 5 μL/min, yielding an approximately13-minute total infusion time. Four weeks following the procedure,animals were sacrificed, and spinal cord tissue was collected foranalyses.

To determine if intraparenchymal administration of the AAV particlesleads to transduction of the spinal cord and knockdown of SOD1 mRNA,ventral horn punches were analyzed by the branched DNA (bDNA) method toquantify levels of SOD1 mRNA, normalized to the geometric mean ofbeta.-actin (ACTB), TATA-box binding protein (TBP) and peptidylprolylisomerase A (PPIA) mRNA levels. These normalized SOD1 mRNA levels werethen expressed relative to normalized. SOD1 mRNA levels in ventral hornpunches from the lumbar region of the spinal cord (L1-L3) from the sameanimals.

Significant SOD1 mRNA knockdown was evident in ventral horn punches fromC1 to T7-10, relative to SOD1 mRNA levels in ventral horn punches fromL1-L3, with similar SOD1 mRNA levels in ventral horn punches from bothsides of the spinal cord. One-way ANOVA and Dunnett's test indicatedsignificant SOD1 mRNA knockdown at each level of the spinal cord (C1-T5p<0.0001; T7-10 p<0.05). As shown in Table 11, spinal cord segmentsclosest to the injections exhibited the greatest SOD1 mRNA knockdown.Spinal segments C1 through C5 had robust and significant knockdown ofSOD1 mRNA (approximately 50-75% knockdown). Even at spinal segment T5,distant from the site of vector injection, significant knockdown of SOD1mRNA (32.6±5.1% knockdown) was observed.

TABLE 11 SOD1 mRNA levels relative to L1-L3 SOD1 mRNA level normalizedto geomean (ACTB, TBP and PPIA) (relative to L1-L3, %) Pig #301 Pig #302Pig #303 Spinal cord Ventral Ventral Ventral Ventral Ventral VentralMean ± segment Horn 1 Horn 2 Horn 1 Horn 2 Horn 1 Horn 2 Standard ErrorC1 47.2 49.9 43.2 48.7 48.3 44.8  47.0 ± 1.0 C2 39.9 41.3 42.3 41.0 49.646.5  43.4 ± 1.5 C3-rostral 25.1 28.2 18.1 15.0 22.6 32.5  23.6 ± 2.6C3-caudal 14.0 17.0 33.1 35.7 29.7 21.3  25.1 ± 3.7 C5-rostral 21.4 19.014.6 21.0 36.1 35.3  24.6 ± 3.7 C5-caudal 25.4 26.6 38.2 33.8 31.7 32.8 31.4 ± 2.0 C7 31.9 15.6 53.6 48.9 44.1 39.2  38.9 ± 5.6 C8 50.1 45.150.5 48.7 36.7 42.8  45.7 ± 2.2 T1-T2 54.0 77.7 55.6 56.0 55.3 55.2 59.0 ± 3.8 T5 65.3 53.1 63.0 58.0 84.9 80.2  67.4 ± 5.1 T7-T10 84.681.5 83.1 68.0 94.3 93.1  84.1 ± 3.9 L1-L3 98.2 93.8 102.0 96.8 105.2103.9 100.0 ± 1.8

Normalized SOD1 mRNA levels in ventral horn punches from AAVparticle-treated pigs were also expressed relative to normalized SOD1mRNA levels in ventral horn punches from the spinal cord of a singlenaive pig. SOD1 mRNA levels were normalized to the geometric mean ofbeta-actin (ACTB), TATA-box binding protein (TBP) and peptidylprolylisomerase A (PPIA) mRNA levels. SOD1 mRNA levels from each cervicalsegment of the treated pigs were then expressed relative to normalizedSOD1 mRNA levels using C2 SOD1 mRNA levels from the nave pig. ThoracicSOD1 mRNA levels (treated pigs) were normalized using T2 SOD1 mRNAlevels (nave pig), and lumbar SOD1 mRNA levels (treated pigs) werenormalized using L2 SOD1 mRNA levels from the nave pig. Ventral hornpunches from the naive pig spinal cord were collected from C2, T2 and L2levels. As shown in Table 12, SOD1 mRNA levels in the ventral hornpunches of the AAV particle administered pigs showed significantknockdown relative to the nave pig (one-way ANOVA and Dunnett's test;p<0.0001) at all spinal cord levels tested. Similar SOD1 mRNA levelswere observed in ventral horn punches from both sides of the spinalcord. SOD1 mRNA knockdown was strongest near the C3 and C5 injectionsites (79-84% knockdown) Even at spinal cord levels distant from thesites of AAV injection, ventral horn punches exhibited significant SOD1mRNA knockdown. At the T5, T7-T10, and L1 spinal cord levels, ventralhorn punches showed significant 55.1±3.4%, 44.0±2,6% and 33.4±1.2%knockdown of SOD1 mRNA, respectively.

TABLE 12 SOD1 mRNA levels relative to naïve control SOD1 mRNA levelnormalized to geomean (ACTB, TBP and PPIA) (relative to naïve control,%) Pig #301 Pig #302 Pig #303 Ventral Ventral Ventral Ventral VentralVentral Spinal cord Horn Horn Horn Horn Horn Horn Mean ± segment Punch 1Punch 2 Punch 1 Punch 2 Punch 1 Punch 2 Standard Error C1 31.5 33.2 28.832.4 32.1 29.8 31.3 ± 0.7 C2 26.6 27.5 28.2 27.3 33.0 30.9 28.9 ± 1.0C3-rostral 16.7 18.8 12.1 10.0 15.0 21.6 15.7 ± 1.8 C3-caudal 9.3 11.322.0 23.8 19.8 14.2 16.7 ± 2.4 C5-rostral 14.3 12.6 9.7 14.0 24.0 23.516.4 ± 2.4 C5-caudal 16.9 17.7 25.5 22.5 21.1 21.8 20.9 ± 1.3 C7 21.210.4 35.7 32.6 29.3 26.1 25.9 ± 3.7 C8 33.3 30.0 33.6 32.4 24.5 28.530.4 ± 1.4 T1-2 36.0 51.7 37.0 37.3 36.8 36.8 39.3 ± 2.5 T5 43.5 35.341.9 38.6 56.5 53.4 44.9 ± 3.4 T7-T10 56.4 54.2 55.3 45.2 62.8 62.0 56.0± 2.6 L1 65.4 62.5 67.9 64.5 70.1 69.2 66.6 ± 1.2

As shown in Table 13, the analysis of vector genome biodistribution bydigital droplet PCR showed high vector genome copy number per diploidcell in ventral horn punches of the cervical spinal cord nearest theinjection sites. Vector genome copy numbers dropped steeply (>10-fold)from C3 to C2, and from C7 to C8 spinal cord levels. However, even atspinal cord levels distant from the C3 and C5 sites of AAV injection,ventral horn punches exhibited significant vector genome copies. At theT5, T7-T10, and L1-L3 spinal cord levels, ventral horn punches showedsignificant 1.7±1.2, 0.2±0.0, and 0.5±0.2 vector genome copies perdiploid cell, respectively.

TABLE 13 Vector Genome Quantification Vector Genome/Diploid Cell (vg/dc)Pig #301 Pig #302 Pig #303 Ventral Ventral Ventral Ventral VentralVentral Spinal cord Horn Horn Horn Horn Horn Horn Mean ± segment Punch 1Punch 2 Punch 1 Punch 2 Punch 1 Punch 2 Standard Error C1 4.4 4.8 10.77.2 4.7 3.1 5.8 ± 1.1 C2 14.1 6.6 25.4 32.1 19.8 13.2 18.5 ± 3.8 C3-rostral 763.1 1305.8 618.1 62.2 798.4 286.2 638.9 ± 177.3 C3-caudal147.3 837.6 1445.7 79.5 185.4 817.1 585.4 ± 221.0 C5-Rostral 677.7 448.01703.8 41.1 70.5 138.2 513.2 ± 278.7 C5-Caudal 644.3 60.7 564.6 70.278.0 174.7 265.4 ± 109.0 C7 29.4 1225.7 5.5 6.1 24.4 9.3 216.7 ± 201.8C8 12.0 22.4 4.2 1.7 7.7 11.6 9.9 ± 3.0 T1-T2 6.7 0.4 1.3 1.6 3.3 1.92.5 ± 0.9 T5 0.6 7.7 0.6 0.5 0.4 0.2 1.7 ± 1.2 T7-T10 0.3 0.3 0.3 0.40.1 0.2 0.2 ± 0.0 L1-L3 0.5 0.4 0.3 0.1 1.4 0.2 0.5 ± 0.2

Vector genome distribution showed a linear correlation to levels of SOD1mRNA knockdown in both analyses, i.e., when SOD1 knockdown was comparedto L1-L3 (r²=0.26, p<0.0001) and when compared to nave control (r²=0.26,p<0.0001). Low vector genome copy number per diploid cell (<1 vg/dc)such as 0.2 or 0.5 vector genome copies per diploid cell on average,still yielded substantial SOD1 mRNA knockdown.

In a second experiment, six Gottingen adult (>9 months of age) femaleand male mini-pigs weighing 15-30 kg each were utilized. Animals werenot pre-screened for neutralizing antibodies to AAV. A multi-levellaminectomy was perfonned at the C3 to C5 levels to access the spinalcord, allowing for 3 cm between injections. Self-complementary (sc) AAVvectors (scAAV) with ITR to ITR sequence of SEQ ID NO: 9, including anH1 promoter and modulatory polynucleotide (SEQ ID NO: 6) comprisingsiRNA targeting SOD1 were packaged in AAVrh10.

In the first group of three pigs, two injections of the scAAV (titer2.03×10¹³ vg/mL) were administered, for a total doselanimal of 1.6×10¹²vg. At the rostral end of the laminectomy, a single 40 μL (8.1×10¹¹ vg)volume was injected into the ventral horn at rostral C3 on the leftside. At the caudal end of the laminectomy, a single 40 μL (8.1E11vg)volume was injected into the ventral horn at caudal C5 on the rightside. Both injections were administered at the rate of 5 μL/min,yielding an approximately 16-minute total infusion time. In the secondgroup of three pigs, vehicle was injected with the same dosing paradigm.Four weeks following the procedure, animals were sacrificed, and spinalcord tissue was collected for analyses.

Ventral horn punches were analyzed by the branched DNA (bDNA) method forknockdown of SOD1 mRNA, normalized to the geometric mean of beta-actin(ACTB), TATA-box binding protein (TBP) and peptidylprolyl isomerase A(PPIA) mRNA levels, and expressed relative to normalized SOD1 mRNAlevels in ventral horn punches from the same spinal cord level ofvehicle treated animals, Significant SOD1 mRNA knockdown was evident inpunches taken from the left ventral horn from C1 to T12 and in punchestaken from the right ventral horn from C1 to L1, with similar SOD1 mRNAlevels in ventral horn punches from both sides of the spinal cord.Two-way ANOVA and Sidak's multiple comparisons test indicatedsignificant SOD1 mRNA knockdown at each level of the spinal cordrelative to the vehicle control group (left side: C1-T7 p<0.0001; T10p<0.001, T12 P<0.01; right side: C1-T10 p<0.0001; T12 p<0.001, L1P<0.01). As shown in Table 14, spinal cord segments closest to theinjections exhibited the greatest SOD1 mRNA knockdown, with the maximalSOD1 mRNA knockdown at C5. Spinal segments C1 through T5 had robust andsignificant knockdown of SOD1 mRNA (50-82% knockdown). Even at spinalsegment T12 on the left side and at spinal cord segment L1 at the rightside, distant from the site of vector injection, significant knockdownof SOD1 mRNA (35.22±2.76%; 29.14±10.36% knockdown, respectively) wasobserved.

TABLE 14 SOD1 mRNA levels relative to vehicle group SOD1 mRNA levelnormalized to geometric mean of housekeeping genes ACTB, TBP and PPIA(relative to vehicle control, %) Left Ventral Horn Vehicle AAV SpinalMean ± Mean ± cord Pig Pig Pig Standard Pig Pig Pig Standard Segments#1001 #1002 #1003 Error #1005 #1004 #1006 Error C1 101.66 91.48 106.86100 ± 4.52  38.45 32.18 37.13 35.92 ± 1.91 C2 79.26 103.29 117.45 100 ±11.15 27.49 30.11 35.06 30.88 ± 2.22 C3 110.32 107.20 82.48 100 ± 8.81 35.71 41.30 34.96 37.32 ± 2.00 C5-Rostral 98.87 122.26 78.86 100 ± 12.5411.33 14.71 28.73 18.26 ± 5.33 C5-Caudal 99.80 91.21 108.99 100 ± 5.13 21.56 20.63 15.97 19.39 ± 1.73 C7 99.00 96.50 104.49 100 ± 2.36  22.5523.66 26.87 24.36 ± 1.30 C8 106.34 92.34 101.31 100 ± 4.09  23.27 32.1129.59 28.32 ± 2.63 T1 89.95 92.54 117.51 100 ± 8.78  30.34 41.09 35.2835.57 ± 3.11 T4 84.33 100.11 115.56 100 ± 9.02  40.53 45.11 41.78 42.47± 1.37 T5 100.90 97.81 101.30 100 ± 1.10  44.22 43.50 41.29 43.00 ± 0.88T7 91.75 102.39 105.85 100 ± 4.24  63.00 49.46 49.49 53.98 ± 4.51 T1088.91 111.54 99.55 100 ± 6.54  62.41 50.33 61.55 58.10 ± 3.89 T12 87.58107.32 105.09 100 ± 6.24  60.37 64.13 69.85 64.78 ± 2.76 L1 102.65104.40 92.95 100 ± 3.56  65.43 58.89 107.53 77.29 ± 15.2 Right Ventralhorn Vehicle AAV Spinal Mean ± Mean ± cord Pig Pig Pig Standard Pig PigPig Standard Segments #1001 #1002 #1003 Error #1005 #1004 #1006 Error C193.18 91.40 115.42 100 ± 7.73  56.06 33.77 41.78 43.87 ± 6.52 C2 100.8391.11 108.07 100 ± 4.91  31.20 31.88 39.79 34.29 ± 2.76 C3 115.93 96.4287.65 100 ± 8.36  27.03 18.23 33.33 26.20 ± 4.38 C5-Rostral 81.86 99.31118.83 100 ± 10.68 23.50 13.53 30.29 22.44 ± 4.87 C5-Caudal 106.72 92.42100.86 100 ± 4.15  32.02 18.18 19.09 23.10 ± 4.47 C7 108.37 88.48 103.15100 ± 5.95  31.51 27.44 24.66 27.87 ± 1.99 C8 107.49 86.23 106.29 100 ±6.90  23.05 26.39 33.22 27.55 ± 2.99 T1 87.95 96.86 115.19 100 ± 8.02 46.68 38.91 37.15 40.91 ± 2.93 T4 93.02 99.40 107.57 100 ± 4.21  41.2241.82 47.36 43.47 ± 1.96 T5 90.86 99.08 110.06 100 ± 5.56  55.96 45.6942.32 47.99 ± 4.10 T7 89.52 99.61 110.87 100 ± 6.17  52.97 51.09 53.8452.63 ± 0.81 T10 98.17 92.35 109.47 100 ± 5.03  55.06 54.18 61.05 56.76± 2.16 T12 86.00 102.54 111.46 100 ± 7.46  57.66 55.25 70.35 61.09 ±4.69 L1 96.12 94.31 109.57 100 ± 4.81  67.49 54.85 90.26 70.86 ± 10.3

As shown in Table 15, the analysis of vector genome biodistribution bydigital droplet PCR showed high vector genome copy number per diploidcell in ventral horn punches of the cervical spinal cord nearest theinjection sites. Vector genome copy numbers dropped steeply (>10-fold onaverage) from C3 to C2, and from C5 to C7 spinal cord levels. However,even at spinal cord levels distant from the C3 and C5 sites of AAVinjection, ventral horn punches exhibited significant vector genomecopies. At the T5, T7, T10, T12, and L1 spinal cord levels, ventral hornpunches showed significant 0.73+0.18, 0.35+0.03, 0.27+0.04, 0.25+0.03,and 0.38+0.19 vector genome copies per diploid cell, respectively.

TABLE 15 Vector Genome Quantification Vector Genome/Dioloid Cell (vg/dc)Left Ventral Horn Right Ventral Horn Spinal Mean ± Mean ± Cord Pig PigPig Standard Pig Pig Pig Standard Segments #1005 #1004 #1006 Error #1005#1004 #1006 Error C1 1.74 3.02 1.16  1.97 ± 0.55 2.59 2.12 1.29 2.00 ±0.38 C2 9.57 9.73 4.52  7.94 ± 1.71 10.69 14.29 4.42 9.80 ± 2.88 C329.66 35.55 27.98 31.06 ± 2.29 585.67 633.71 28.65 416.01 ± 194.17C5-Rostral 92.67 187.13 439.19  239.66 ± 103.42 45.37 201.47 554.95267.27 ± 150.74 C5-Caudal 3029.44 760.00 332.53 1373.99 ± 836.88 132.37960.73 290.71 461.27 ± 253.88 C7 18.39 28.38 56.81  34.53 ± 11.51 9.5227.43 41.11 26.02 ± 9.14  C8 5.15 6.82 11.99  7.98 ± 2.06 3.57 10.3715.65 9.86 ± 3.50 T1 2.03 4.03 7.06  4.37 ± 1.46 2.66 4.40 5.83 4.30 ±0.92 T4 0.51 0.52 0.84  0.63 ± 0.11 0.63 1.15 0.95 0.91 ± 0.15 T5 0.431.54 0.58  0.85 ± 0.35 0.35 0.92 0.55 0.60 ± 0.17 T7 0.23 0.42 0.31 0.32 ± 0.06 0.40 0.41 0.32 0.38 ± 0.03 T10 0.13 0.27 0.36  0.25 ± 0.070.26 0.36 0.24 0.29 ± 0.04 T12 0.21 0.17 0.28  0.22 ± 0.03 0.27 0.220.38 0.29 ± 0.05 L1 1.32 0.16 0.12  0.53 ± 0.39 0.25 0.30 0.13 0.22 ±0.05

Vector genome distribution showed linear correlation to levels of SOD1mRNA knockdown, when compared to vehicle control (r²=0.15, p=0.0002).50% SOD1 KD was achieved with low vector genome copy number per diploidcell (>1 vg/dc) such as 0.2 or 0.5 vector genome copies per diploid cellon average, in ventral horn punches ˜30 cm caudal to the injection site.

Equivalents and Scope

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or the entiregroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anyantibiotic, therapeutic or active ingredient; any method of production;any method of use; etc.) can he excluded from any one or more claims,for any reason, whether or not related to the existence of prior art.

It is to be understood that the words which have been used are words ofdescription rather than limitation, and that changes may be made withinthe purview of the appended claims without departing from the true scopeand spirit of the invention in its broader aspects.

While the present invention has been described at some length and withsome particularity with respect to the several described embodiments, itis not intended that it should he limited to any such particulars orembodiments or any particular embodiment, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the invention.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

Section and table headings are not intended to be limiting.

We claim:
 1. A method for inhibiting the expression of the superoxidedismutase 1 (SOD1) gene in a subject comprising administering to thesubject a composition comprising an adeno-associated viral (AAV) vectorcomprising a vector genome and a capsid; wherein said vector genomecomprises a modulatory polynucleotide sequence positioned between twoinverted terminal repeats (ITRs) and wherein administration occurs atone or more sites via intraparenchymal delivery.
 2. The method of claim1, wherein administration occurs at one or more sites within the spinalcord of the subject.
 3. The method of claim 2, wherein administrationoccurs at two sites within the spinal cord of the subject.
 4. The methodof claim 3, wherein administration occurs at two sites at the cervicalspinal cord region.
 5. The method of claim 4, wherein the two sites atthe cervical spinal cord region are at levels C3 and C5.
 6. The methodof claim 4, wherein the two sites at the cervical spinal cord region areat two levels selected from the group consisting of C1, C2, C3, C4, C5,C6, and C7.
 7. The method of claim 3, wherein administration occurs attwo sites at the thoracic spinal cord region.
 8. The method of claim 7,wherein the two sites at the thoracic spinal cord region are at twolevels selected from the group consisting of T1, T2, T3, T4, T5, T6, T7,T8, T9, T10, T11, and T12.
 9. The method of claim 3, whereinadministration occurs at two sites at the lumbar spinal cord region. 10.The method of claim 9, wherein the two sites at the lumbar spinal cordregion are at two levels selected from the group consisting of L1, L2,L3, L4 and L5.
 11. The method of claim 2 or 3, wherein administrationoccurs at one or more sites located at one or more regions independentlyselected from the group consisting of cervical spinal cord, thoracicspinal cord, lumbar spinal cord, and sacral spinal cord.
 12. The methodof claim 11, wherein the one or more sites are independently selectedfrom the group consisting of C1, C2, C3, C4, C5, C6, C7, T1, T2, T3, T4,T5, T6, T7, T8, T9, T10, T11, T12, L1, L2, L3, L4, and L5.
 13. Themethod of claim 11, wherein the one or more sites are independentlyselected from the group consisting of C1, C2, C3, C4, C5, C6, C7, andL
 1. 14. A method for treating and/or ameliorating amyotrophic lateralsclerosis (ALS) in a subject, the method comprising administering to thesubject in need of treatment a therapeutically effective amount of acomposition comprising an adeno-associated viral (AAV) vector comprisinga vector genome and a capsid; wherein said vector genome comprises amodulatory polynucleotide sequence positioned between two invertedterminal repeats (ITR) and wherein administration occurs at one or moresites by intraparenchymal delivery.
 15. The method of claim 14, whereinthe expression of SOD1 is inhibited or suppressed.
 16. The method of anyone of claims 14-15, wherein the SOD1 is wild type SOD1, mutated SOD1with at least one mutation or both wild type SOD1 and mutated SOD1 withat least one mutation.
 17. The method of any one of claims 14-16,wherein the expression of SOD1 is inhibited or suppressed by about 20%to about 100%.
 18. The method of claims 14-17, wherein administrationoccurs at one or more sites within the spinal cord of the subject. 19.The method of claim 18, wherein administration occurs at two siteswithin the spinal cord of the subject.
 20. The method of claim 19.wherein administration occurs at two sites at the cervical spinal cordregion.
 21. The method of claim 20, wherein the two sites at thecervical spinal cord region are at levels C3 and C5.
 22. The method ofclaim 21, wherein the volume of administration is 5 μL to 240 μL atlevel C3 of the spinal cord and 5 μL to 240 μL at level C5 of the spinalcord.
 23. The method of claim 21, wherein the volume of administrationis 5 μL to 60 μL at level C3 of the spinal cord and 5 μL, to 60 μL atlevel C5 of the spinal cord.
 24. The method of claim 21, wherein thevolume of administration is 25 to 40 μL at level C3 of the spinal cordand 25 to 40 μL at level C5 of the spinal cord.
 25. The method of claim21, wherein the dose is 1×10¹⁰ vg to 1×10¹² vg at level C3 of the spinalcord and 1×10¹⁰ vg to 1×10¹² vg at level C5 of the spinal cord.
 26. Themethod of claim 21, wherein the dose is 5×10¹¹ vg to 8×10¹¹ vg at levelC. the spinal cord and 5×10¹¹ vg to 8×10¹¹ vg at level C5 of the spinalcord.
 27. The method of any one of claims 22-24, wherein the compositionis administered at a rate of 5 μL/min.
 28. The method of claim 20,wherein the two sites at the cervical spinal cord region are at twolevels selected from the group consisting of C1, C2, C3, C4, C5, C6, andC7.
 29. The method of claim 19, wherein administration occurs at twosites at the thoracic spinal cord region.
 30. The method of claim 29,wherein the two sites at the thoracic spinal cord region are at twolevels selected from the group consisting of T1, T2, T3, T4, T5, T6, T7,T8, T9, T10, T11, and T12.
 31. The method of claim 19, whereinadministration occurs at two sites at the lumbar spinal cord region. 32.The method of claim 31, wherein the two sites at the lumbar spinal cordregion are at two levels selected from the group consisting of L1, L2,L3, L4, and L5.
 33. The method of claim 18 or 19, wherein administrationoccurs at one or more sites located at one or more regions selected fromthe group consisting of cervical spinal cord, thoracic spinal cord,lumbar spinal cord, and sacral spinal cord.
 34. The method of claim 33,wherein the one or more sites are independently selected from the groupconsisting of C1, C2, C3, C4, C5, C6, C7, T1, T2, T3, T4, T5, T6, T7,T8, T9, T10, T11, T12, L1, L2, L3, L4, and L5.
 35. The method of claim33, wherein the one or more sites are independently selected from thegroup consisting of C1, C2, C3, C4, C5, C6, C7. and L1.
 36. The methodof any one of claims 1-35, wherein the modulatory polynucleotidesequence comprises a sense strand sequence and an antisense strandsequence, wherein the sense strand sequence comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from thenucleotide sequence of SEQ ID NO: 7 and the antisense strand sequencecomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO: 8 and whereinsaid sense strand sequence and antisense strand sequence share a regionof complementarily of at least four nucleotides in length.
 37. Themethod of claim 36, wherein the modulatory polynucleotide sequencecomprises a sense strand sequence and an antisense strand sequence of ansiRNA duplex,
 38. The method of claim 37, wherein the siRNA duplex isD-4012.
 39. The method of claim 36, wherein the region ofcomplementarity is at least 17 nucleotides in length.
 40. The method ofclaim 39, wherein the region of complementarity is between 19 and 21nucleotides in length.
 41. The method of claim 39, wherein the region ofcomplementarity is 19 nucleotides in length.
 42. The method of any oneof claims 36-41, wherein the sense strand sequence and the antisensestrand sequence are, independently, 30 nucleotides or less.
 43. Themethod of any one of claims 36-42, wherein at least one of the sensestrand sequence and the antisense strand sequence comprise a 3′ overhangof at least 1 nucleotide.
 44. The method of claim 43, wherein at leastone of the sense strand sequence and the antisense strand sequencecomprise a 3′ overhang of at least 2 nucleotides.
 45. The method of anyone of claims 1-44, wherein the capsid is a serotype selected. from thegroup consisting of AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3,AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8,AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47NAV9.61,AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1,AAV27.3, AAV42.12, AAV42-1h, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4,AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12,AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21,AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1,AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7/rh.48,AAV1-8/rh,49, AAV2-15/rh.62, AAV2-3/rh.61, AAV2-4/rh.50, AAV2-5/rh.51,AAV3.1/hu.6, AAV3.1/hu.9, AAV3-9/fh.52, AAV3-11./rh.53, AAV4-8/r11.64,AAV4-9/rh.54, AAV4-19/rh.55, AAV5-3/rh.57, AAV5-22.1rh.58,AAV7.3/hu,7AAV16.8/hu.10, AAV16.12/hu.11, AAV29.3/bb.1, AAV29.5/bb.2,AAV106.1/hu.37, AAV114.3/hu.40, AAV127.2/hu.41, AAV127.5/hu.42,AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1 /hu.53, AAV145.5/hu.54,AAV145.6/hu.55, AAV161.10/hu.60, AAV 161 .6/hu.61, AAV33.12/hu.17,AAV33.4/hu.15, AAV33.8/hu.16, AAV52/hu.19, AAV52.1/hu.20, AAV58.2/hu.25,AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAVC1, AAVC2, AAVC5, AAV-DJ,AAV-DJ8, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70,AAVpi.1, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55,AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVLK03,AAVH-1/hu.1, AAVH-5/hu.3, AAVLG-10/rh.40, AAVLG-4/rh.38, AAVLG-9/hu.39,AAVN721-8/rh.43, AAVCh.5, AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5,AAVCy.5R1, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.1, AAVhu.2,AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10,AAVhu.11, AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.20,AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28,AAVhu.29, AAVhu.29R AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37,AAVhu.39. AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44R1,AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48,AAVhu.48R1, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52,AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61,AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14/9, AAVhu.t19, AAVrh.2,AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R,AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22,AAVrh,23, AAVrh,24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34,AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40,AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2NAVrh.48.2, AAVrh.49,AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58,AAVrh,61AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74,AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, AAAV, BAAV, caprineAAV, bovine AAV, AAVhE1.1, AAVhEr1.5, AAVhER114, AAVhEr1.8, AAVhEr1.16,AAVhEr1.18, AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29AAVhEr2.4,AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36AAVhER1.23, AAVhEr3.1,AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05,AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12,AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, ANV-LK18NAV-LK19,AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11,AAV-PAEC12, AAV-2-pre-miRNA-101 AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM10-2 AAV Shuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAVShuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAVSM 10-1, AAV SM 10-8 , AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19,AAVhu.11, AAVhu.53, AAV4-8/fh.64, AAVLG-9/hu.39, AAV54.5/hu.23,AAV54.2/hu.22, AAV54.7/hu.24, AAV54.1/hu.21, AAV54.4R/hu.27,AAV46.2/hu.28, AAV46.6/hu.29, AAV128.1/hu.43, true type AAV (ttAAV),UPENN AAV 10, Japanese AAV 10 serotypes, AAV CBr-7.1, AAV CBr-7.10, AAVCBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAVCBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-E1, AAV CBr-E2, AAV CBr-E3,AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8,AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5,AAV CHt-6.6AAV CHt-6.7, AAV CHt-6.8 AAV CHt-P1, AAV CHt-P2, AAV CHt-P5,AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2,AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-B1, AAVCKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAVCKd-B8, AAV CKd-H1, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKA-H5, AAVCKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-F1, AAV CLg-F2,NAVCLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAVCLv-1, AAV CLv1-1, AAV Clv1-10, AAV CLv1-2, AAV CLv-12, AAV CLv1-3, AAVCLv-13, AAV CLv1-4, AAV Clv1-7, AAV Clv1-8, AAV Clv1-9, AAV CLv-2, AAVCLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-D1, AAV CLv-D2, AAVCLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAVCLv-E1, AAV CLv-K1, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAVCLv-L6, AAV CLv-M1, AAV CLv-M11, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAVCLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-R1, AAV CLv-R2, AAV CLv-R3, AAVCLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAVCSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAVCSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.10, AAV CSp-8.2, AAV CSp-8.4, AAVCSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9,AAV.hu.48R3, AAV,VR-355, AAV3B, AAV4, AAV5, AAVF1/HSC1, AAVF11/HSC11,AAVF12/HSC12, AAVF13/HISC13, AAVF14/HSC14, AAVF15/HSC15, AAVF16/HSC16,AAVF17/HSC17, AAVF2/HSC2, AAVF3/HSC3, AAVF4/HSC4, AAVF5/HSC5,AAVF6/HSC6, AAVF7/HSC7, AAVF8/HSC8, AAVF9/HSC9, AAV-PHP.B, AAV-PHP.A,G2B-26, G2B-13, TH1.1-32, TH1.1-35, AAVPHP.B2, AAVPHP.B3,AAVPHP.N/PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP,AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, A.AVPHP.B-GGT-T, AAVPHP.B-SGS,AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP,AAVPHIP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT,AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP,AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S/G2A12, AAVG2A15/G2A3,AAVG2B4, AAVG2B5 and variants thereof.
 46. The method of claim 45,wherein the capsid is AAVrh.10 serotype or a variant thereof.
 47. Themethod of any one of claims 1-46, wherein the AAV vector comprises apromoter, and wherein the promoter is H1.